**4. Philadelphia chromosome positive acute lymphoblastic leukemia (Ph + ALL)**

indicating that IM could be tolerated for longer period of time post-transplant with no major hematological drawbacks. However, peripheral blood counts should be monitored during the duration of treatment for pancytopenia, a leading side effect of TKIs [37]. A study by DeAngelo et al. evaluated 15 patients who received IM for relapse post-transplant, 10 of whom were in CP, one in AP, and four patients in BP [34]. Nine of the ten patients who received IM in CP achieved a complete cytogenetic response after six months. The OS rate was at one 100% after 25 months of follow-up. Kantarjian et al. studied 28 CML relapse patient post-SCT [32], all of whom received IM in a dose range of 400–1000 mg/day. Five patients were in CP, 15 in AP, and 8 in BP. Thirteen patients received DLI at an average of 4 months prior to the use of IM. Complete hematologic response was seen in 100% of the CP patients, 83% in AP, and 43% in BP. The OS was 74% after 1 year of follow-up. The study concluded that no difference was detected in complete response between patients who had received IM and DLI, in comparison to patients who received IM alone. A study by Hess et al. evaluated 44 patients, 37 of whom were in CP before SCT, 18 patients had molecular relapse, while 19 had cytogenetic relapse [38]. IM started post-SCT on an average of 2.1 years yielded a complete molecular response in 62% of the patients after 9 months that further improved with subsequent follow-ups. They also recommended the use of molecular end point in clinical decision making. On the other hand, a study by Olavarria et al. showed that standard PCR techniques were lacking; however, complete molecular response had been significantly high, especially in CP patients [31]. In another study by Olavarria et al., IM was administrated 35 days postrelapse in 22 patients. In a year, all patients had achieved complete molecular remission without cytogenetic relapse during the entire length of IM therapy [33]. Relapse had only been detected after the discontinuation of IM therapy. The length of IM use post-transplant is yet to be determined. These various studies have recommended IM as a feasible, effective, and well-tolerated treatment for relapsing CML patients after SCT. Currently, there is a paucity of data to determine the efficacy of using TKI after SCT for maintenance. However, given its efficacy in relapsed disease, many

The available data about the use of second generation TKI, such as dasatinib, is still limited [39]. A study by Klyuchnikov et al. used second-generation TKI dasatinib post HST in 11 patients; out of whom nine had CML, two were in AP, and seven in BP [40]. All the patients had received TKI prior to their SCT-IM, dasatinib, or nilotinib (sometimes in combination). Dasatinib was administrated at a median interval of 1 year post-SCT with a median duration of treatment of 8 months (it was discontinued in one patient due to gastrointestinal bleeding that was thought due to drug-related thrombocytopenia). Responses to dasatinib posttransplant were seen in four out of the nine patients. Five patients failed to respond to dasatinib, while three passed away due to disease progression. One patient had a CNS relapse. The study concluded that second-generation TKI dasatinib is effective in the treatment of relapse of CML post-transplant and is generally well tolerated despite the hematological side effects. Contrary

to IM, dasatinib was able to penetrate extramedullary tissues and CNS [41, 42].

physicians use this strategy for maintenance.

200 Stem Cells in Clinical Practice and Tissue Engineering

Before the use of allogeneic SCT for the treatment of Philadelphia chromosome positive Acute Lymphoblastic Leukemia (Ph + ALL), patients were usually treated with induction chemotherapy alone. It had poor outcomes in regard to the long-term survival with a median diseasefree survival (DFS) range of 5–9 months [43]. SCT has been used as a curative treatment for Ph + ALL postinduction chemotherapy to improve the long-term survival [44]. One such retrospective study compared patients treated with chemotherapy alone, with patients being treated with chemotherapy followed by SCT [45]. The study reported significant survival improvement in patients treated with chemotherapy followed by SCT than patients treated with chemotherapy alone.

Detection of BCR-ABL post-SCT using RT-PCR was found to be a good predictor of minimal residual disease (MRD). Relapse with RT-PCR turned positive 4–6 months prior to the occurrence of relapse in patients who achieved remission after chemotherapy with and without combination with SCT [46].

In a study by Chen et al., the effects of TKI IM on DFS post-SCT in patients with Ph + ALL, 82 patients were evaluated of which 62 patients had received IM at a median of 70 days post-SCT for a median duration of 90 days. In 14 patients, BCR-ABL was positive prior to the use of IM, while 8 patients turned negative after a 1 month use of IM. Relapse rate was 10.2% in the group using IM, while it was 33.1% in non-IM group. The 5-year DFS was 81.5% in IM group in comparison to 33.5% in the non-IM group. Multivariate analysis proved the use of IM post-SCT as a prognostic factor for DFS and OS (*P* = 0.000) [47]. Twenty-two patients were studied by Carpenter et al. to assess the tolerance of IM use post-SCT [48]: 15 patients with Ph + ALL and seven patients with CML. IM was easily tolerated by 17 out of the 19 adult patients at doses of 400 mg/day. In the pediatric age group, it was tolerated by three children at doses of 260 mg/m2 /day in the first 90 days post-SCT (doses compared with those used in primary therapy for Ph + leukemia). Fourteen patients had positive BCR-ABL before SCT. After an average 1.4-year follow-up, 17 patients were alive with negative BCR-ABL transcripts. In the Ribera et al. study, 30 patients newly diagnosed Ph + ALL received IM with chemotherapy followed by SCT [49]. Transplant was then followed by IM for a median duration of 3.9 months. Twenty-seven patients achieved remission of which 21 patients underwent SCT. Twelve patients received IM after transplant. After a follow-up of 4.1 years, the DFS and the OS were 30 and 30%, respectively. Contrary to previous studies, Ribera et al. showed that adverse effects due to transplant limited the early use of IM post-SCT despite the efficacy of combined IM and chemotherapy as a primary treatment for Ph + ALL.

In another study by Teng et al., a similar TKI, dasatinib was evaluated for the same purpose [50]. Six patients with Ph + ALL were enrolled in the study and received SCT along with 100 mg/ day dasatinib for 1 year. All patients achieved complete remission prior to SCT with relapse occurring in three patients only (all extramedullary). Only two patients had adverse effects from dasatinib which was improved by dividing the dose of dasatinib into two 50 mg doses. This study showed that dasatinib was effective and tolerated by Ph + ALL patients after SCT.

### **5. Acute myeloid leukemia (AML)**

Although SCT is considered as a treatment for acute myeloid leukemia (AML) to either induce remission, or to prevent relapse, relapse is perhaps considered to be the most common complication post-SCT in patients with AML especially in the first year after transplant [51]. The duration of remission after SCT is the most important predictor of for survival after relapse [52]. Like ALL, the detection of MRD using RT-PCR could predict the post-transplant relapse in patents with AML and is much more accurate than other classic methods [53]. The 5 years OS was 79% in MRD-negative patients as detected by flow cytometry as opposed to 26% in MRD-positive patients with relapse rates of 58 and 14% after 2 years, respectively [54]. Several modalities have been studied to help control post-transplant relapse in AML patients. Jabbour et al. studied the efficacy and feasibility of Azacitidine (a DNA hypomethylating agent) in nine patients to prevent disease recurrence after SCT and in eight patients as maintenance therapy [55]. It was administrated daily for 5 days and was repeated every 4 weeks for a median duration of follow-up of 11 months. The EFS and OS rates after a year were 55 and 90%, respectively. In a paper by Cruijsen et al., 27 patients with AML who had relapsed post-SCT received Azacitidine for 3 days at a total daily dose of 100 mg which was followed by donor lymphocyte infusion on day 10 [56]. The next course started on day 22. A total of 60 courses of Azacitidine were administered. The OS from the initiation of Azacitidine was 136 days (range of 23–873), and the survival rate after 2 years was 16%. In a study by Bolanos-Meade et al., 10 patients with AML relapse post-SCT receiving Azacitidine 75 mg/m2 / day for 5–7 days were evaluated [57]. Of the 10, six achieved complete remission, one had stable disease, and three progressed after a median of six cycles. After a median follow-up of 624 days, the OS was 422.5 days (range 127–1411). These studies demonstrated that Azacitidine is effective and well tolerated by the patients without exacerbation of GVHD.

administered for 5 days in a 6 weeks cycle. The median follow-up post-HSCT was 24 months. Approximately 43% of the patients were able to receive all eight cycles. It was determined that a dose of 15 mg/m2 administered for 5 days, every 6 weeks was safe. Although results were not statistically significant, an increasing FOXP3 expression was observed in all patients. The lack of toxicities and low GVHD incidence indicated that further studies had to be con-

A more recent and similar study by Pusic et al. [59], DAC was assessed for its safety and efficacy as a maintenance therapy in patients with AML and MDS post-HSCT. Twenty-two patients were enrolled and divided into four cohorts. DAC was administered in four doses (5, 7.5, 10,

completed all eight cycles out of which eight patients remained in CR. DAC maintenance did not clearly impact the rate of chronic GVHD, although a similar trend of increased FOXP3 expression seen. DACs maintenance was associated with acceptable toxicities when given in the postallogenic HSCT setting. Although the maximum tolerated dose was not reached, the

for 5 days every 6 weeks appeared to be the optimal dose.

Sorafenib is a tyrosine kinase inhibitor (TKI) that inhibits the FLT3 tyrosine kinase receptor. The FLT3-ITD mutation is associated with a high relapse rates for patients with AML postallogenic HSCT. Chen et al. [60] conducted a Phase 1 trial using sorafenib as maintenance therapy for post-HSCT in patients with FLT3-ITD AML. The patients received an assortment of conditioning regimens (10 reduced intensity and 12 myeloablative). They were all in morphological remission with predominant donor chimerism post-HSCT before starting maintenance therapy. A dose escalation 3 + 3 cohort design was used to define the maximum tolerated dose (MTD). Ten patients were additionally treated at the MTD. Sorafenib was started between days 45 and 120 post-HSCT and given continuously for 12 cycles, each cycle consisting of 28 days. No significant flares of acute GVHD was observed after starting sorafenib. The 1 year cumulative incidence of chronic GVHD was 42% (90% CI, 23%, 60%). Serial FLT3 ligand levels were measured in seven patients. Median level at baseline and prior to any drug administration was 125 pg/ml, which significantly increased to a median level of 254 pg/ml (*P* = 0.016) measured on day 29 of cycle 1. The surviving patient median follow-up was 14.5 months post-HSCT. The 1 year PFS is 84% (90% CI, 63–94%) and 1 year OS is 95% (90% CI, 79–99%). Only one patient relapsed post-HSCT. The study concluded Sorafenib may reduce the rate of relapse, was safe to give as maintenance therapy after HSCT for patients with FLT3-ITD AML. The use of maintenance sorafenib and other FLT3 inhibitors post-HSCT warrants further investigation.

The use of high-dose chemotherapy followed by ASCT has been in use for a long time as a potential treatment for diffuse large B cell lymphoma (DLBCL). However, it is now mostly restricted to chemosensitive DLBCL [61, 62]. The PARMA trial began with 216 patients who received two courses of DHAP (dexamethasone, Ara-C, cisplatin) confirmed the superiority of dose intensification with autologous bone marrow transplantation over conventional

/day), for 5 days every 6 weeks, for a maximum of eight cycles. Nine patients

Post-Transplantation Management Strategies

http://dx.doi.org/10.5772/65239

203

ducted to determine the exact dosage of DAC required for maintenance.

and 15 mg/m2

dose of 10 mg/m2

**6. Non Hodgkins Lymphoma**

A Phase II trial was conducted by De Lima et al. to determine the maintenance dose of azacitidine postallogenic HSCT for recurrent AML or MDS [58]. Forty-five high risk patients with a median age of 60 years were treated. A combination of five different azacitidine was investigated (8, 16, 24, 32, and 40 mg/m2 ), on four different schedules. Each scheduled cycle consisted of 5 days of drug administration and 25 days of rest. Reversible thrombocytopenia was found out to be the dose limiting toxicity. The optimal azacitidine combination determined from this study was 32 mg/m2 administered for four cycles with a median follow-up of 20.5 months. The 1-year event-free survival and OS were 58 and 77%, respectively.

Decitabine (DAC) is a hypomethylating agent that irreversibly binds and inhibits DNA methyltransferase-1, resulting in loss of DNA methylation. Using DAC in maintenance therapy may help eradicate minimal residual disease and facilitate a graft versus leukemia effect by enhancing the effect of T-regulatory lymphocytes. One of the first studies to use Decitabine (DAC) as a maintenance drug postallogenic HSCT in pretreated patients with AML and MDS was conducted in 2012 by Choi et al. [59]. A total of 19 patients with a median age of 60 years were enrolled out of which 14 had AML, and five patients had MDS. All conditioning regimens were myeloablative. Three cohorts had been completed and a final fourth cohort is currently enrolling. Four doses of DAC had been investigated (5, 7.5, 10, and 15 mg/m2 /day) administered for 5 days in a 6 weeks cycle. The median follow-up post-HSCT was 24 months. Approximately 43% of the patients were able to receive all eight cycles. It was determined that a dose of 15 mg/m2 administered for 5 days, every 6 weeks was safe. Although results were not statistically significant, an increasing FOXP3 expression was observed in all patients. The lack of toxicities and low GVHD incidence indicated that further studies had to be conducted to determine the exact dosage of DAC required for maintenance.

A more recent and similar study by Pusic et al. [59], DAC was assessed for its safety and efficacy as a maintenance therapy in patients with AML and MDS post-HSCT. Twenty-two patients were enrolled and divided into four cohorts. DAC was administered in four doses (5, 7.5, 10, and 15 mg/m2 /day), for 5 days every 6 weeks, for a maximum of eight cycles. Nine patients completed all eight cycles out of which eight patients remained in CR. DAC maintenance did not clearly impact the rate of chronic GVHD, although a similar trend of increased FOXP3 expression seen. DACs maintenance was associated with acceptable toxicities when given in the postallogenic HSCT setting. Although the maximum tolerated dose was not reached, the dose of 10 mg/m2 for 5 days every 6 weeks appeared to be the optimal dose.

Sorafenib is a tyrosine kinase inhibitor (TKI) that inhibits the FLT3 tyrosine kinase receptor. The FLT3-ITD mutation is associated with a high relapse rates for patients with AML postallogenic HSCT. Chen et al. [60] conducted a Phase 1 trial using sorafenib as maintenance therapy for post-HSCT in patients with FLT3-ITD AML. The patients received an assortment of conditioning regimens (10 reduced intensity and 12 myeloablative). They were all in morphological remission with predominant donor chimerism post-HSCT before starting maintenance therapy. A dose escalation 3 + 3 cohort design was used to define the maximum tolerated dose (MTD). Ten patients were additionally treated at the MTD. Sorafenib was started between days 45 and 120 post-HSCT and given continuously for 12 cycles, each cycle consisting of 28 days. No significant flares of acute GVHD was observed after starting sorafenib. The 1 year cumulative incidence of chronic GVHD was 42% (90% CI, 23%, 60%). Serial FLT3 ligand levels were measured in seven patients. Median level at baseline and prior to any drug administration was 125 pg/ml, which significantly increased to a median level of 254 pg/ml (*P* = 0.016) measured on day 29 of cycle 1. The surviving patient median follow-up was 14.5 months post-HSCT. The 1 year PFS is 84% (90% CI, 63–94%) and 1 year OS is 95% (90% CI, 79–99%). Only one patient relapsed post-HSCT. The study concluded Sorafenib may reduce the rate of relapse, was safe to give as maintenance therapy after HSCT for patients with FLT3-ITD AML. The use of maintenance sorafenib and other FLT3 inhibitors post-HSCT warrants further investigation.

### **6. Non Hodgkins Lymphoma**

/

/day)

**5. Acute myeloid leukemia (AML)**

202 Stem Cells in Clinical Practice and Tissue Engineering

investigated (8, 16, 24, 32, and 40 mg/m2

from this study was 32 mg/m2

Although SCT is considered as a treatment for acute myeloid leukemia (AML) to either induce remission, or to prevent relapse, relapse is perhaps considered to be the most common complication post-SCT in patients with AML especially in the first year after transplant [51]. The duration of remission after SCT is the most important predictor of for survival after relapse [52]. Like ALL, the detection of MRD using RT-PCR could predict the post-transplant relapse in patents with AML and is much more accurate than other classic methods [53]. The 5 years OS was 79% in MRD-negative patients as detected by flow cytometry as opposed to 26% in MRD-positive patients with relapse rates of 58 and 14% after 2 years, respectively [54]. Several modalities have been studied to help control post-transplant relapse in AML patients. Jabbour et al. studied the efficacy and feasibility of Azacitidine (a DNA hypomethylating agent) in nine patients to prevent disease recurrence after SCT and in eight patients as maintenance therapy [55]. It was administrated daily for 5 days and was repeated every 4 weeks for a median duration of follow-up of 11 months. The EFS and OS rates after a year were 55 and 90%, respectively. In a paper by Cruijsen et al., 27 patients with AML who had relapsed post-SCT received Azacitidine for 3 days at a total daily dose of 100 mg which was followed by donor lymphocyte infusion on day 10 [56]. The next course started on day 22. A total of 60 courses of Azacitidine were administered. The OS from the initiation of Azacitidine was 136 days (range of 23–873), and the survival rate after 2 years was 16%. In a study by Bolanos-Meade et al., 10 patients with AML relapse post-SCT receiving Azacitidine 75 mg/m2

day for 5–7 days were evaluated [57]. Of the 10, six achieved complete remission, one had stable disease, and three progressed after a median of six cycles. After a median follow-up of 624 days, the OS was 422.5 days (range 127–1411). These studies demonstrated that Azacitidine is

A Phase II trial was conducted by De Lima et al. to determine the maintenance dose of azacitidine postallogenic HSCT for recurrent AML or MDS [58]. Forty-five high risk patients with a median age of 60 years were treated. A combination of five different azacitidine was

consisted of 5 days of drug administration and 25 days of rest. Reversible thrombocytopenia was found out to be the dose limiting toxicity. The optimal azacitidine combination determined

Decitabine (DAC) is a hypomethylating agent that irreversibly binds and inhibits DNA methyltransferase-1, resulting in loss of DNA methylation. Using DAC in maintenance therapy may help eradicate minimal residual disease and facilitate a graft versus leukemia effect by enhancing the effect of T-regulatory lymphocytes. One of the first studies to use Decitabine (DAC) as a maintenance drug postallogenic HSCT in pretreated patients with AML and MDS was conducted in 2012 by Choi et al. [59]. A total of 19 patients with a median age of 60 years were enrolled out of which 14 had AML, and five patients had MDS. All conditioning regimens were myeloablative. Three cohorts had been completed and a final fourth cohort is currently enrolling. Four doses of DAC had been investigated (5, 7.5, 10, and 15 mg/m2

20.5 months. The 1-year event-free survival and OS were 58 and 77%, respectively.

), on four different schedules. Each scheduled cycle

administered for four cycles with a median follow-up of

effective and well tolerated by the patients without exacerbation of GVHD.

The use of high-dose chemotherapy followed by ASCT has been in use for a long time as a potential treatment for diffuse large B cell lymphoma (DLBCL). However, it is now mostly restricted to chemosensitive DLBCL [61, 62]. The PARMA trial began with 216 patients who received two courses of DHAP (dexamethasone, Ara-C, cisplatin) confirmed the superiority of dose intensification with autologous bone marrow transplantation over conventional chemotherapy in patients with relapsed diffuse NHL [62]. In different types of lymphoma, relapse is the most important cause of mortality post-transplant, particularly within the first 9 months post-SCT. This is further demonstrated in a study conducted by Hamdani et al., where patients with DLBCL were compared among autologous HCT outcomes for chemosensitive DLBCL patients between 2000 and 2011 [63]. These were divided in two cohorts based on time to relapse from diagnosis. The early rituximab failure (ERF) cohort consisted of patients with primary refractory disease or patients who had first relapse within a year of their initial diagnosis. This group was then compared with those patients who had relapses more than a year after initial diagnosis (late rituximab failure [LRF] cohort). Both the ERF and LRF cohorts included 300 and 216 patients, respectively. Nonrelapse mortality (NRM), OS, PFS values of ERF versus LRF groups at the 3 years were 9% (95% confidence interval [CI], 6–13%) versus 9% (95% CI, 5–13%, 50% (95% CI, 44–56%) versus 67% (95% CI, 60–74%), and 44% (95% CI, 38– 50%) versus 52% (95% CI, 45–59%), respectively. On a multivariate analysis, the ERF was not associated with a higher NRM (relative risk [RR], 1.31; *P* = 0.34). The ERF group experienced a higher risk of treatment failure (RR, 2.08; *P* < 0.001) and overall mortality (RR, 3.75; *P* < 0.001) within the first 9 months after autologous HCT. Beyond this period, the PFS and OS were not significantly different between both groups of LRF and ERF. Several studies have evaluated different treatment approaches for relapse post-SCT. In one such study by Haioun et al., 269 patients randomized into two groups receive rituximab maintenance (*n* = 139) for four weeks, or observed without maintenance (*n* = 130) after SCT [64]. Patients were then randomized into two groups of those achieving complete response (CR) (*n* = 130), and those who achieved incomplete or partial response (*n* = 139). After a median follow-up of 4 years from the second randomization, the EFS was 80% in the rituximab arm in comparison to 70% in observation only arm with no statistically significant difference in between both groups (*P* = 0.99), though significant difference was found in both arms of patients who achieved CR post-SCT. In another study by Gisselbrecht et al., of the 477 relapsed patients enrolled, 242 responded to salvage therapy and received SCT and high-dose chemotherapy [65]. They were then assigned to receive either rituximab for 1 year (*n* = 122), or observation alone (*n* = 120). After a median 44 months of follow-up post-SCT, no significant difference was found regarding EFS, PFS, or OS between both groups. Interestingly, significant difference was found in EFS between women (63%) and men (46%) in the rituximab group. This could be explained by the higher concentration of rituximab in females due to their slower body release [66]. The quality of life of 269 patients with DLBCL randomized to receive either rituximab or observation alone post-SCT was done in a study conducted by Heutte et al. [67]. The study showed that Rituximab decreased pain and severity of symptoms, with the long-term difference in quality of life post-SCT was not dependent on rituximab maintenance. As concluded by these studies, rituximab could be used as a maintenance therapy post-SCT as being a feasible and safe option, but does not improve disease control or survival outcome and needs to be investigated further.

complete response of 145 patients with SCT, 36 had molecular relapse after a mean of 18.5 months following SCT, and 26 patients got administrated pre-emptive rituximab which could induce a remission of 92%. Median clinical and molecular-free survival was 3.7 and 1.5 years, respectively, stating the importance of PCR analysis for patients with MCL to stratify

Post-Transplantation Management Strategies

http://dx.doi.org/10.5772/65239

205

As in non-Hodgkin lymphoma, high-dose therapy followed by autologous stem-cell transplantation is the standard of care for relapsed patients of Hodgkin's lymphoma (HL), and for patients who did not respond to the salvage treatment [74]. Studies have shown that SCT could significantly increase the progression-free survival (PFS) [75]. Several treatment approaches have been studied to increase outcome after SCT as radiation before and after SCT and consolidation post-SCT [76, 77]. Brentuximab vedotin, an anti-CD 30 antibody linked to protease cleavage agent has been studied as a post-transplant therapy for HL. A Phase II study by Younes et al. showed that response rate to brentuximab is 75% in relapsed patients with HL and complete response rate was 34% after 2 years [78]. The AETHERA study [79] evaluated brentuximab as an early consolidation therapy post-SCT where 329 patients with relapsed or refractory HL were randomly assigned to receive 16 cycles of 1.8 mg/kg brentuximab vedotin (*n* = 165), or placebo (*n* = 164) starting 30–45 days after SCT. PFS was significantly higher in patients in the brentuximab vedotin group compared with those in the placebo group (95% CI 0.40–0.81; *P* = 0.0013), with median PFS of 42.5 months in brentuximab receiving patients compared with 24.1 months in patients receiving placebo. After 24 months of follow-up, 63% of brentuximab group were alive in comparison with the 51% in the placebo group. The study concluded that the administration of brentuximab early after SCT in relapsed or refractory HL

IMiDs have also decreased relapse rates along with a decrease in the PFS and OS rates; increasing the mean survival from the previous 3-year survival to an 8-year post-transplant survival. The combination of thalidomide and steroid is promising and has shown significant improvement in the EFS and PF rates in comparison to a lone steroid therapy, although neuropathy is still a major concern in thalidomide-based regimen for prolonged use. Lenalidomide surpasses in tolerance when compared with thalidomide due to its unique efficacy and toxicity profile and has proven to be an effective maintenance therapy following AHSCT with a significant impact on improving the PFS and OS. Similarly, Bortezomib has its defined efficacy and toxicity profile and showed significant increase in PFS and OS in patients as a

*MM summary*: IMiDs have decreased relapse rates and PFS OS rates. Mean survival with the

maintenance therapy in renal-impaired patients suffering from MM.

new modalities is 8 years post-transplant from 3 years.

high-risk group of patients.

**7. Hodgkins Lymphoma**

patients had significantly improved EFS and OS.

**8. Conclusion**

Though mantle cell lymphoma (MCL) is still considered a poor prognosis type of non-Hodgkin lymphoma [68], rituximab has proven efficacy in Phase III studies by prolonging disease-free survival and improving clinical response in patients with MCL undergone SCT [69]. Better response was correlated with detection of PCR undetectable markers in bone marrow and peripheral blood after SCT [70–72]. In a study by Andersen et al. [73], 74 patients showed complete response of 145 patients with SCT, 36 had molecular relapse after a mean of 18.5 months following SCT, and 26 patients got administrated pre-emptive rituximab which could induce a remission of 92%. Median clinical and molecular-free survival was 3.7 and 1.5 years, respectively, stating the importance of PCR analysis for patients with MCL to stratify high-risk group of patients.

### **7. Hodgkins Lymphoma**

chemotherapy in patients with relapsed diffuse NHL [62]. In different types of lymphoma, relapse is the most important cause of mortality post-transplant, particularly within the first 9 months post-SCT. This is further demonstrated in a study conducted by Hamdani et al., where patients with DLBCL were compared among autologous HCT outcomes for chemosensitive DLBCL patients between 2000 and 2011 [63]. These were divided in two cohorts based on time to relapse from diagnosis. The early rituximab failure (ERF) cohort consisted of patients with primary refractory disease or patients who had first relapse within a year of their initial diagnosis. This group was then compared with those patients who had relapses more than a year after initial diagnosis (late rituximab failure [LRF] cohort). Both the ERF and LRF cohorts included 300 and 216 patients, respectively. Nonrelapse mortality (NRM), OS, PFS values of ERF versus LRF groups at the 3 years were 9% (95% confidence interval [CI], 6–13%) versus 9% (95% CI, 5–13%, 50% (95% CI, 44–56%) versus 67% (95% CI, 60–74%), and 44% (95% CI, 38– 50%) versus 52% (95% CI, 45–59%), respectively. On a multivariate analysis, the ERF was not associated with a higher NRM (relative risk [RR], 1.31; *P* = 0.34). The ERF group experienced a higher risk of treatment failure (RR, 2.08; *P* < 0.001) and overall mortality (RR, 3.75; *P* < 0.001) within the first 9 months after autologous HCT. Beyond this period, the PFS and OS were not significantly different between both groups of LRF and ERF. Several studies have evaluated different treatment approaches for relapse post-SCT. In one such study by Haioun et al., 269 patients randomized into two groups receive rituximab maintenance (*n* = 139) for four weeks, or observed without maintenance (*n* = 130) after SCT [64]. Patients were then randomized into two groups of those achieving complete response (CR) (*n* = 130), and those who achieved incomplete or partial response (*n* = 139). After a median follow-up of 4 years from the second randomization, the EFS was 80% in the rituximab arm in comparison to 70% in observation only arm with no statistically significant difference in between both groups (*P* = 0.99), though significant difference was found in both arms of patients who achieved CR post-SCT. In another study by Gisselbrecht et al., of the 477 relapsed patients enrolled, 242 responded to salvage therapy and received SCT and high-dose chemotherapy [65]. They were then assigned to receive either rituximab for 1 year (*n* = 122), or observation alone (*n* = 120). After a median 44 months of follow-up post-SCT, no significant difference was found regarding EFS, PFS, or OS between both groups. Interestingly, significant difference was found in EFS between women (63%) and men (46%) in the rituximab group. This could be explained by the higher concentration of rituximab in females due to their slower body release [66]. The quality of life of 269 patients with DLBCL randomized to receive either rituximab or observation alone post-SCT was done in a study conducted by Heutte et al. [67]. The study showed that Rituximab decreased pain and severity of symptoms, with the long-term difference in quality of life post-SCT was not dependent on rituximab maintenance. As concluded by these studies, rituximab could be used as a maintenance therapy post-SCT as being a feasible and safe option, but does not improve disease control or survival outcome and needs to be investigated further.

204 Stem Cells in Clinical Practice and Tissue Engineering

Though mantle cell lymphoma (MCL) is still considered a poor prognosis type of non-Hodgkin lymphoma [68], rituximab has proven efficacy in Phase III studies by prolonging disease-free survival and improving clinical response in patients with MCL undergone SCT [69]. Better response was correlated with detection of PCR undetectable markers in bone marrow and peripheral blood after SCT [70–72]. In a study by Andersen et al. [73], 74 patients showed As in non-Hodgkin lymphoma, high-dose therapy followed by autologous stem-cell transplantation is the standard of care for relapsed patients of Hodgkin's lymphoma (HL), and for patients who did not respond to the salvage treatment [74]. Studies have shown that SCT could significantly increase the progression-free survival (PFS) [75]. Several treatment approaches have been studied to increase outcome after SCT as radiation before and after SCT and consolidation post-SCT [76, 77]. Brentuximab vedotin, an anti-CD 30 antibody linked to protease cleavage agent has been studied as a post-transplant therapy for HL. A Phase II study by Younes et al. showed that response rate to brentuximab is 75% in relapsed patients with HL and complete response rate was 34% after 2 years [78]. The AETHERA study [79] evaluated brentuximab as an early consolidation therapy post-SCT where 329 patients with relapsed or refractory HL were randomly assigned to receive 16 cycles of 1.8 mg/kg brentuximab vedotin (*n* = 165), or placebo (*n* = 164) starting 30–45 days after SCT. PFS was significantly higher in patients in the brentuximab vedotin group compared with those in the placebo group (95% CI 0.40–0.81; *P* = 0.0013), with median PFS of 42.5 months in brentuximab receiving patients compared with 24.1 months in patients receiving placebo. After 24 months of follow-up, 63% of brentuximab group were alive in comparison with the 51% in the placebo group. The study concluded that the administration of brentuximab early after SCT in relapsed or refractory HL patients had significantly improved EFS and OS.

### **8. Conclusion**

IMiDs have also decreased relapse rates along with a decrease in the PFS and OS rates; increasing the mean survival from the previous 3-year survival to an 8-year post-transplant survival. The combination of thalidomide and steroid is promising and has shown significant improvement in the EFS and PF rates in comparison to a lone steroid therapy, although neuropathy is still a major concern in thalidomide-based regimen for prolonged use. Lenalidomide surpasses in tolerance when compared with thalidomide due to its unique efficacy and toxicity profile and has proven to be an effective maintenance therapy following AHSCT with a significant impact on improving the PFS and OS. Similarly, Bortezomib has its defined efficacy and toxicity profile and showed significant increase in PFS and OS in patients as a maintenance therapy in renal-impaired patients suffering from MM.

*MM summary*: IMiDs have decreased relapse rates and PFS OS rates. Mean survival with the new modalities is 8 years post-transplant from 3 years.

*Thalidomide summary*: thalidomide + dexamethasone usage has shown good improvement in EFS and PF rates compared with using dexamethasone alone. OS values are variable. Neuropathy is the major concern in thalidomide-based regimen for long-term use. No optimal duration for thalidomide has been established.

**Conflict of interest**

**Author details**

**References**

Muhammad Waqas Khan1\*, Ahmed Elmaaz2

University of Kentucky, Kentucky, USA

Med. 2003 May 8;**348**(19):1875-83

Clin Oncol. 1999;**17**(1):208-215

ma. Blood. 2010;**115**(6):1113-1120

2012;**119**(1):7-15

The authors have no conflict of interest, nor have received any funding.

\*Address all correspondence to: mkh233@uky.edu and gulzh@ucmail.uc.edu

1 Department of Internal Medicine division of Hematology and Bone marrow transplant,

2 Department of Hematology, Oncology, University of Cincinnati, Cincinnati, Ohio, USA

[1] Attal M, Harousseau JL, Facon T, Guilhot F, Doyen C, Fuzibet JG, Monconduit M, Hulin C, Caillot D, Bouabdallah R, Voillat L, Sotto JJ, Grosbois B, Bataille R; InterGroupe Francophone du Myélome. Single versus double autologous stem-cell transplantation

[2] Child JA, Morgan GJ, Davies FE, Owen RG, Bell SE, Hawkins K, Brown J, Drayson MT, Selby PJ; Medical Research Council Adult Leukaemia Working Party. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J

[3] Barlogie B, Tricot GJ, Van Rhee F, et al. Long-term outcome results of the first tandem autotransplant trial for multiple myeloma. Br J Haematol. 2006;**135**(2):158-164

[4] Corradini P, Voena C, Tarella C, et al. Molecular and clinical remissions in multiple myeloma: role of autologous and allogeneic transplantation of hematopoietic cells. J

[5] Morgan GJ, Gregory WM, Davies FE, et al. The role of maintenance thalidomide therapy in multiple myeloma: MRC Myeloma IX results and meta-analysis. Blood.

[6] Lokhorst HM, van der Holt B, Zweegman S, et al. A randomized phase 3 study on the effect of thalidomide combined with adriamycin, dexamethasone, and high-dose melphalan, followed by thalidomide maintenance in patients with multiple myelo-

for multiple myeloma. N Engl J Med. 2003 Dec 25;**349**(26):2495-502

and Zartash Gul1\*

Post-Transplantation Management Strategies

http://dx.doi.org/10.5772/65239

207

*Lenalidomide summary*: due to its higher efficacy and a lower toxicity profile the use of lenalidomide surpasses in tolerance when compared with thalidomide. Lenalidomide is an effective maintenance therapy following AHSCT, having and has significant impact on improving the PFS and OS. Long-term usage was found to be associated with an elevated risk of hematologic complications, neutropenia. SPM risk was significantly higher in patients receiving melphalan + lenalidomide.

*Bortezomib summary*: bortezomibs has a defined efficacy and toxicity profile. Significant increase in PFS and OS in patients using bortezomib post-HSCT. Bortezomib is effective as a maintenance therapy in renal impaired patients suffering from MM. Exact dose of maintenance therapy is not yet known.

*CML summary*: TKIs have proven to improve survival following SCT. The efficacy of TKIs following SCT depends on the phase of the disease; a good prognosis if TKIs were used for relapse CP phase, less if used for relapse in the AP or BP phases. Dasatinib is effective in the treatment of relapse of CML post-transplant, is well tolerated despite the hematological side effects. Dasatinib can penetrate extramedullary tissues and CNS. Presently, there is an insufficiency of current data required to determine the efficacy of using TKI after SCT for maintenance. Nonetheless, given its efficacy in relapsed disease, many physicians use this strategy for maintenance.

*Ph* + *ALL summary*: SCT has been used as a curative treatment for Ph + ALL postinduction chemotherapy to improve the long-term survival. Dasatinib was effective and tolerated by Ph + ALL patients after SCT.

*AML summary*: the duration of remission after SCT is the most important predictor of for survival after relapse. Azacitidine, decitabine, sorafenib, and other FLT3 inhibitors are effective and well tolerated by the patients without exacerbation of GVHD. Although studies of using FLT3 inhibitors as maintenance therapies are still on going, the data collected so far shows promising results and merits further trials.

*NHL summary*: high-dose chemotherapy followed by ASCT has been in use for a long time as a potential treatment for DLBCL. A 5-year event-free survival rate was significantly higher in patients who received ASCT and chemotherapy, than patients receiving salvage therapy alone. Rituximab could be used as a maintenance therapy post-SCT as being a feasible and safe option, but does not improve disease control or survival outcome and needs to be investigated.

*HL summary*: SCT could significantly increase the progression-free survival. Brentuximab early after SCT in relapsed or refractory HL patients had significantly improved EFS and OS.

### **Conflict of interest**

*Thalidomide summary*: thalidomide + dexamethasone usage has shown good improvement in EFS and PF rates compared with using dexamethasone alone. OS values are variable. Neuropathy is the major concern in thalidomide-based regimen for long-term use. No optimal

*Lenalidomide summary*: due to its higher efficacy and a lower toxicity profile the use of lenalidomide surpasses in tolerance when compared with thalidomide. Lenalidomide is an effective maintenance therapy following AHSCT, having and has significant impact on improving the PFS and OS. Long-term usage was found to be associated with an elevated risk of hematologic complications, neutropenia. SPM risk was significantly higher in patients receiving melpha-

*Bortezomib summary*: bortezomibs has a defined efficacy and toxicity profile. Significant increase in PFS and OS in patients using bortezomib post-HSCT. Bortezomib is effective as a maintenance therapy in renal impaired patients suffering from MM. Exact dose of maintenance

*CML summary*: TKIs have proven to improve survival following SCT. The efficacy of TKIs following SCT depends on the phase of the disease; a good prognosis if TKIs were used for relapse CP phase, less if used for relapse in the AP or BP phases. Dasatinib is effective in the treatment of relapse of CML post-transplant, is well tolerated despite the hematological side effects. Dasatinib can penetrate extramedullary tissues and CNS. Presently, there is an insufficiency of current data required to determine the efficacy of using TKI after SCT for maintenance. Nonetheless, given its efficacy in relapsed disease, many physicians use this

*Ph* + *ALL summary*: SCT has been used as a curative treatment for Ph + ALL postinduction chemotherapy to improve the long-term survival. Dasatinib was effective and tolerated by

*AML summary*: the duration of remission after SCT is the most important predictor of for survival after relapse. Azacitidine, decitabine, sorafenib, and other FLT3 inhibitors are effective and well tolerated by the patients without exacerbation of GVHD. Although studies of using FLT3 inhibitors as maintenance therapies are still on going, the data collected so far shows

*NHL summary*: high-dose chemotherapy followed by ASCT has been in use for a long time as a potential treatment for DLBCL. A 5-year event-free survival rate was significantly higher in patients who received ASCT and chemotherapy, than patients receiving salvage therapy alone. Rituximab could be used as a maintenance therapy post-SCT as being a feasible and safe option, but does not improve disease control or survival outcome and needs to be investigated.

*HL summary*: SCT could significantly increase the progression-free survival. Brentuximab early after SCT in relapsed or refractory HL patients had significantly improved EFS and OS.

duration for thalidomide has been established.

206 Stem Cells in Clinical Practice and Tissue Engineering

lan + lenalidomide.

therapy is not yet known.

strategy for maintenance.

Ph + ALL patients after SCT.

promising results and merits further trials.

The authors have no conflict of interest, nor have received any funding.

### **Author details**

Muhammad Waqas Khan1\*, Ahmed Elmaaz2 and Zartash Gul1\*

\*Address all correspondence to: mkh233@uky.edu and gulzh@ucmail.uc.edu

1 Department of Internal Medicine division of Hematology and Bone marrow transplant, University of Kentucky, Kentucky, USA

2 Department of Hematology, Oncology, University of Cincinnati, Cincinnati, Ohio, USA

### **References**


[7] Barlogie B, Pineda-Roman M, van Rhee F, et al. Thalidomide arm of total therapy 2 improves complete remission duration and survival in myeloma patients with metaphase cytogenetic abnormalities. Blood. 2008;**112**(8):3115-3121

((Delta)13) in multiple myeloma: an Eastern Cooperative Oncology Group study. Cancer

Post-Transplantation Management Strategies

http://dx.doi.org/10.5772/65239

209

[21] Spencer A, Prince HM, Roberts AW, et al. Consolidation therapy with low-dose thalidomide and prednisolone prolongs the survival of multiple myeloma patients undergoing a single autologous stem-cell transplantation procedure. J Clin Oncol.

[22] Maiolino A, Hungria VT, Garnica M, et al. Thalidomide plus dexamethasone as a maintenance therapy after autologous hematopoietic stem cell transplantation improves progression-free survival in multiple myeloma. Am J Hematol. 2012;**87**(10):

[23] Dredge K, Marriott JB, Todryk SM, et al. Protective antitumor immunity induced by a costimulatory thalidomide analog in conjunction with whole tumor cell vaccination is

mediated by increased Th1-type immunity. J Immunol. 2002;**168**(10):4914-4919

[24] Richardson PG, Schlossman RL, Weller E, et al. Immunomodulatory drug CC-5013 overcomes drug resistance and is well tolerated in patients with relapsed multiple

[25] Attal M, Roussel M. Maintenance therapy for myeloma: how much, how long, and at

[26] McCarthy PL, Hahn T. Strategies for induction, autologous hematopoietic stem cell transplantation, consolidation, and maintenance for transplantation-eligible multiple myeloma patients. Hematology Am Soc Hematol Educ Program. 2013;**2013**:496-503.

[27] Palumbo A, Bringhen S, Kumar SK, et al. Second primary malignancies with lenalidomide therapy for newly diagnosed myeloma: a meta-analysis of individual patient

[28] Abidi MH, Gul Z, Abrams J, et al. Phase I trial of bortezomib during maintenance phase after high dose melphalan and autologous stem cell transplantation in patients with

[29] Horowitz MM, Rowlings PA, Passweg JR. Allogeneic bone marrow transplantation for CML: a report from the International Bone Marrow Transplant Registry. Bone Marrow

[30] Dazzi F, Szydlo RM, Cross NC, et al. Durability of responses following donor lymphocyte infusions for patients who relapse after allogeneic stem cell transplantation for

[31] Olavarria E, Ottmann OG, Deininger M, et al. Response to imatinib in patients who relapse after allogeneic stem cell transplantation for chronic myeloid leukemia. 

Res. 2002;**62**(3):715-720

2009;**27**(11):1788-1793

myeloma. Blood. 2002;**100**(9):3063-3067

doi:10.1182/asheducation-2013.1.496

data. Lancet Oncol. 2014;**15**(3):333-342

Transplant. 1996;**17**(Suppl 3):S5-S6

Leukemia. 2003;**17**(9):1707-1712

multiple myeloma. J Chemother. 2012 Jun;**24**(3)

chronic myeloid leukemia. Blood. 2000;**96**(8):2712-2716

what cost? Am Soc Clin Oncol Educ Book. 2012:515-522

948-952


((Delta)13) in multiple myeloma: an Eastern Cooperative Oncology Group study. Cancer Res. 2002;**62**(3):715-720

[21] Spencer A, Prince HM, Roberts AW, et al. Consolidation therapy with low-dose thalidomide and prednisolone prolongs the survival of multiple myeloma patients undergoing a single autologous stem-cell transplantation procedure. J Clin Oncol. 2009;**27**(11):1788-1793

[7] Barlogie B, Pineda-Roman M, van Rhee F, et al. Thalidomide arm of total therapy 2 improves complete remission duration and survival in myeloma patients with meta-

[8] Stewart AK, Chen CI, Howson-Jan K, et al. Results of a multicenter randomized phase II trial of thalidomide and prednisone maintenance therapy for multiple myeloma after

[9] Attal M, Harousseau J-L, Leyvraz S, et al. Maintenance therapy with thalidomide improves survival in patients with multiple myeloma. Blood. 2006;**108**(10):3289-3294

[10] Attal M, Lauwers-Cances V, Marit G, et al. Lenalidomide maintenance after stem-cell transplantation for multiple myeloma. N Engl J Med. 2012;**366**(19):1782-1791

[11] Alsina M, Becker PS, Zhong X, et al. Lenalidomide maintenance for high-risk multiple myeloma after allogeneic hematopoietic cell transplantation. Biol Blood Marrow

[12] McCarthy PL, Owzar K, Hofmeister CC, et al. Lenalidomide after stem-cell transplan-

[13] Kneppers E, van der Holt B, Kersten M-J, et al. Lenalidomide maintenance after nonmyeloablative allogeneic stem cell transplantation in multiple myeloma is not

[14] Rosiñol L, Oriol A, Teruel AI, et al. Superiority of bortezomib, thalidomide, and dexamethasone (VTD) as induction pretransplantation therapy in multiple myeloma:

[15] Sonneveld P, Schmidt-Wolf IGH, van der Holt B, et al. Bortezomib induction and maintenance treatment in patients with newly diagnosed multiple myeloma: results of the randomized phase III HOVON-65/ GMMG-HD4 trial. J Clin Oncol. 2012;**30**(24):

[16] Scheid C, Sonneveld P, Schmidt-Wolf IGH, et al. Bortezomib before and after autologous stem cell transplantation overcomes the negative prognostic impact of renal impairment in newly diagnosed multiple myeloma: a subgroup analysis from the

[17] Fritz E, Ludwig H. Interferon-alpha treatment in multiple myeloma: meta-analysis of 30 randomised trials among 3948 patients. Ann Oncol. 2000;**11**(11):1427-1436

[18] Kumar SK, Rajkumar SV, Dispenzieri A, et al. Improved survival in multiple myeloma

[19] Morgan GJ, Davies FE, Gregory WM, et al. Long-term follow-up of MRC myeloma IX trial: Survival outcomes with bisphosphonate and thalidomide treatment. Clin Cancer

[20] Fonseca R, Harrington D, Oken MM, et al. Biological and prognostic significance of interphase fluorescence in situ hybridization detection of chromosome 13 abnormalities

HOVON-65/GMMG-HD4 trial. Haematologica. 2014;**99**(1):148-154

and the impact of novel therapies. Blood. 2008;**111**(5):2516-2520

a randomized phase 3 PETHEMA/GEM study. Blood. 2012;**120**(8):1589-1596

tation for multiple myeloma. N Engl J Med. 2012;**366**(19):1770-1781

feasible: results of the HOVON 76 Trial. Blood. 2011;**118**(9):2413-2419

autologous stem cell transplant. Clin Cancer Res. 2004;**10**(24):8170-8176

phase cytogenetic abnormalities. Blood. 2008;**112**(8):3115-3121

Transplant. 2014;**20**(8):1183-1189

208 Stem Cells in Clinical Practice and Tissue Engineering

2946-2955

Res. 2013;**19**(21):6030-6038


[32] Kantarjian HM, O'Brien S, Cortes JE, et al. Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood. 2002;**100**(5):1590-1595

[44] Snyder DS. Allogeneic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia. Biol Blood Marrow Transplant. 2000;**6**(1083–8791 SB–

Post-Transplantation Management Strategies

http://dx.doi.org/10.5772/65239

211

[45] Arico M, Valsecchi MG, Camitta B, et al. Outcome of treatment in children with Philadelphia chromosome-positive acute lymphoblastic leukemia. N Engl J Med.

[46] Preudhomme C, Henic N, Cazin B, et al. Good correlation between RT-PCR analysis and relapse in Philadelphia (Ph1)-positive acute lymphoblastic leukemia (ALL). 

[47] Chen H, Liu K, Xu L, et al. Administration of imatinib after allogeneic hematopoietic stem cell transplantation may improve disease-free survival for patients with Philadelphia chromosome-positive acute lymphobla stic leukemia. J Hematol Oncol.

[48] Carpenter PA, Snyder DS, Flowers MED, et al. Prophylactic administration of imatinib after hematopoietic cell transplantation for high-risk Philadelphia chromosome-

[49] Ribera J-M, Oriol A, González M, et al. Concurrent intensive chemotherapy and imatinib before and after stem cell transplantation in newly diagnosed Philadelphia chromosome-positive acute lymphoblastic leukemia. Final results of the CSTIBES02

[50] Teng C-LJ, Yu J-T, Chen H-C, Hwang W-L. Maintenance therapy with dasatinib after allogeneic hematopoietic stem cell transplantation in Philadelphia chromosome-

[51] Meijer E, Cornelissen JJ. Allogeneic stem cell transplantation in acute myeloid leukemia in first or subsequent remission: weighing prognostic markers predicting relapse and

[52] Oran B, de Lima M. Prevention and treatment of acute myeloid leukemia relapse after allogeneic stem cell transplantation. Curr Opin Hematol. 2011;**18**(6):388-394

[53] Voskova D, Schoch C, Schnittger S, Hiddemann W, Haferlach T, Kern W. Stability of leukemia-associated aberrant immunophenotypes in patients with acute myeloid leukemia between diagnosis and relapse: comparison with cytomorphologic, cytogenetic, and molecular genetic findings. Cytom Part B—Clin Cytom. 2004;**62**(1):25-38

[54] Walter RB, Gooley TA, Wood BL, et al. Impact of pretransplantation minimal residual disease, as detected by multiparametric flow cytometry, on outcome of myeloablative hematopoietic cell transplantation for acute myeloid leukemia. J Clin Oncol.

[55] Jabbour E, Giralt S, Kantarjian H, et al. Low-dose azacitidine after allogeneic stem cell

transplantation for acute leukemia. Cancer. 2009;**115**(9):1899-1905

positive acute lymphoblastic leukemia. Ann Hematol. 2013;**92**(8):1137-1139

risk factors for non-relapse mortality. Semin Oncol. 2008;**35**(4):449-457

IM):597-603

2012;**5**:29

2000;**342**(14):998-1006

Leukemia. 1997;**11**(0887–6924 (Print)):294-298

positive leukemia. Blood. 2007;**109**(7):2791-2793

trial. Haematologica. 2010;**95**(1):87-95

2011;**29**(9):1190-1197


[44] Snyder DS. Allogeneic stem cell transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia. Biol Blood Marrow Transplant. 2000;**6**(1083–8791 SB– IM):597-603

[32] Kantarjian HM, O'Brien S, Cortes JE, et al. Imatinib mesylate therapy for relapse after allogeneic stem cell transplantation for chronic myelogenous leukemia. Blood.

[33] Olavarria E, Kanfer E, Szydlo R, et al. Early detection of BCR-ABL transcripts by quantitative reverse transcriptase-polymerase chain reaction predicts outcome after allogeneic stem cell transplantation for chronic myeloid leukemia. Blood. 2001;**97**(6):

[34] DeAngelo DJ, Hochberg EP, Alyea EP, et al. Extended follow-up of patients treated with imatinib mesylate (Gleevec) for chronic myelogenous leukemia relapse after allogeneic transplantation: Durable cytogenetic remission and conversion to complete donor chimerism without graft-versus-host disease. Clin Cancer Res. 2004;**10**(15):5065-5071

[35] Druker BJ, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the abl tyrosine kinase on the growth of Bcr-Abl positive cells. Nat Med. 1996;**2**(5):561-566

[36] Wright MP, Shepherd JD, Barnett MJ, et al. Response to tyrosine kinase inhibitor therapy in patients with chronic myelogenous leukemia relapsing in chronic and advanced phase following allogeneic hematopoietic stem cell transplantation. Biol Blood Marrow

[37] Palandri F, Amabile M, Rosti G, et al. Imatinib therapy for chronic myeloid leukemia patients who relapse after allogeneic stem cell transplantation: a molecular analysis. 

[38] Hess G, Bunjes D, Siegert W, et al. Sustained complete molecular remissions after treatment with imatinib-mesylate in patients with failure after allogeneic stem cell transplantation for chronic myelogenous leukemia: results of a prospective phase II

[39] Reddiconto G, Chiusolo P, Fiorina A, et al. Dasatinib restores full donor chimerism in a patient with imatinib-resistant Ph + ALL relapsing after unrelated cord blood

[40] Klyuchnikov E, Schafhausen P, Kröger N, et al. Second-generation tyrosine kinase inhibitors in the post-transplant period in patients with chronic myeloid leukemia or Philadelphia-positive acute lymphoblastic leukemia. Acta Haematol. 2009;**122**(1):6-10

[41] Altintas A, Cil T, Kilinc I, Kaplan MA, Ayyildiz O. Central nervous system blastic crisis in chronic myeloid leukemia on imatinib mesylate therapy: a case report. J Neurooncol.

[42] Aichberger KJ, Herndlhofer S, Agis H, et al. Liposomal cytarabine for treatment of myeloid central nervous system relapse in chronic myeloid leukaemia occurring during

[43] Faderl S, Kantarjian HM, Thomas DA, et al. Outcome of Philadelphia chromosomepositive adult acute lymphoblastic leukemia. Leuk Lymphoma. 2000;**36**:263-273

open-label multicenter study. J Clin Oncol. 2005;**23**(30):7583-7593

transplantation. Leuk Lymphoma. 2015;**48**(10):2054-2057

imatinib therapy. Eur J Clin Invest. 2007;**37**(10):808-813

2002;**100**(5):1590-1595

210 Stem Cells in Clinical Practice and Tissue Engineering

Transplant. 2010;**16**(5):639-646

2007;**84**(1):103-105

Bone Marrow Transplant. 2007;**39**(3):189-191

1560-1565


[56] Cruijsen M, Lübbert M, Wijermans P, et al. Clinical results of hypomethylating agents in AML treatment. J Clin Med. 2015 Jan;**4**(1):1-17

[67] Heutte N, Haioun C, Feugier P, et al. Quality of life in 269 patients with poor-risk diffuse large B-cell lymphoma treated with rituximab versus observation after autologous stem

Post-Transplantation Management Strategies

http://dx.doi.org/10.5772/65239

213

[68] Epperla N, Fenske TS, Hari PN, Hamadani M. Recent advances in post autologous transplantation maintenance therapies in B-cell non-Hodgkin lymphomas. World J

[69] Andersen NS, Jensen MK, de Nully Brown P, Geisler CH. A Danish population-based analysis of 105 mantle cell lymphoma patients: incidences, clinical features, response, survival and prognostic factors. Eur J Cancer. 2002;**38**(3):401-408. doi:10.1016/

[70] Magni M, Di Nicola M, Devizzi L, et al. Successful in vivo purging of CD34-containing peripheral blood harvests in mantle cell and indolent lymphoma: evidence for a role of both chemotherapy and rituximab infusion. Blood. 2000;**96**(3):864-869. doi:10.1016/

[71] Geisler CH, Kolstad A, Laurell A, et al. Long-term progression-free survival of mantle cell lymphoma after intensive front-line immunochemotherapy with in vivo-purged stem cell rescue: a nonrandomized phase 2 multicenter study by the Nordic Lymphoma

[72] Corradini P, Astolfi M, Cherasco C, et al. Molecular monitoring of minimal residual disease in follicular and mantle cell non-Hodgkin's lymphomas treated with high-dose chemotherapy and peripheral blood progenitor cell autografting. Blood. 1997;**89**(2):

[73] Andersen NS, Pedersen LB, Laurell A, et al. Pre-emptive treatment with rituximab of molecular relapse after autologous stem cell transplantation in mantle cell lymphoma.

[74] Moskowitz CH, Nademanee A, Masszi T, et al. Brentuximab vedotin as consolidation therapy after autologous stem-cell transplantation in patients with Hodgkin's lymphoma at risk of relapse or progression (AETHERA): a randomised, double-blind, placebo-

[75] Schmitz N, Pfistner B, Sextro M, et al. Aggressive conventional chemotherapy compared with high-dose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin's disease: A randomised trial. Lancet.

[76] Moskowitz AJ, Perales MA, Kewalramani T, et al. Outcomes for patients who fail high dose chemoradiotherapy and autologous stem cell rescue for relapsed and primary

refractory Hodgkin lymphoma. Br J Haematol. 2009;**146**(2):158-163

controlled, phase 3 trial. Lancet. 2015;**385**(9980):1853-1862

Group. Blood. 2008;**112**(7):2687-2693. doi:10.1182/blood-2008-03-147025

cell transplant. Leuk Lymphoma. 2011;**52**(7):1239-1248

Transplant. 2015;**5**(3):81-88

S0959-8049(01)00366-5

s0889-8588(05)70470-6

J Clin Oncol. 2009;**27**(26):4365-4370

2002;**359**(9323):2065-2071

724-731


[67] Heutte N, Haioun C, Feugier P, et al. Quality of life in 269 patients with poor-risk diffuse large B-cell lymphoma treated with rituximab versus observation after autologous stem cell transplant. Leuk Lymphoma. 2011;**52**(7):1239-1248

[56] Cruijsen M, Lübbert M, Wijermans P, et al. Clinical results of hypomethylating agents

[57] Bolaños-Meade J, Smith BD, Gore SD, et al. 5-azacytidine as salvage treatment in relapsed myeloid tumors after allogeneic bone marrow transplantation. Biol Blood

[58] Choi J, Bernabe N, Abboud CN, et al. Maintenance therapy with decitabine after allogeneic hematopoietic stem cell transplantation for acute myeloid leukemia and

[59] Pusic I, Choi J, Fiala MA, et al. Maintenance therapy with decitabine after allogeneic stem cell transplantation for acute myelogenous leukemia and myelodysplastic

[60] Chen YB, Shuli L, Andrew LA, et al. Phase I trial of maintenance Sorafenib after allogeneic hematopoietic stem cell transplantation for patients with FLT3-ITD AML.

[61] Gianni AM, Bregni M, Siena S, et al. High-dose chemotherapy and autologous bone marrow transplantation compared with MACOP-B in aggressive B-cell lymphoma. N

[62] Philip T, Guglielmi C, Hagenbeek A, et al. Autologous bone marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-

[63] Hamdani M, Hari PN, Zhang Y, et al. Early failure of frontline rituximab-containing chemo-immunotherapy in diffuse large B cell lymphoma does not predict futility of autologous hematopoietic cell transplantation. Biol Blood Marrow Transplant.

[64] Haioun C, Mounier N, Emile JF, et al. Rituximab compared to observation after highdose consolidative first-line chemotherapy (HDC) with autologous stem cell transplantation in poor-risk diffuse large B-cell lymphoma: updated results of the LNH98-

[65] Gisselbrecht C, Schmitz N, Mounier N, et al. Rituximab maintenance therapy after autologous stem-cell transplantation in patients with relapsed CD20+ diffuse large Bcell lymphoma: final analysis of the collaborative trial in relapsed aggressive lympho-

[66] Pfreundschuh M, Poeschel V, Zeynalova S, et al. Optimization of rituximab for the treatment of diffuse large B-cell lymphoma (II): extended rituximab exposure time in the SMARTE-R-CHOP-14 trial of the German high-grade non-Hodgkin lymphoma

in AML treatment. J Clin Med. 2015 Jan;**4**(1):1-17

high-risk myelodysplastic syndrome. Blood. 2013;**122**:4638

Hodgkin's lymphoma. N Engl J Med. 1995;**333**(23):1540-1545

B3 GELA study. J Clin Oncol (Meeting Abstr). 2007;**25**(18\_suppl)

syndrome. Biol Blood Marrow Transplant. 2015 Oct;**21**(10):1761-1769

Marrow Transplant. 2011;**17**(5):754-758

212 Stem Cells in Clinical Practice and Tissue Engineering

Blood. 2014;**124**:671

2014;**20**(11):1729-1736

Engl J Med. 1997;**336**(18):1290-1297

ma. J Clin Oncol. 2012;**30**(36):4462-4469

study group. J Clin Oncol. 2014;**32**(36):4127-4133


[77] Rapoport AP, Guo C, Badros A, et al. Autologous stem cell transplantation followed by consolidation chemotherapy for relapsed or refractory Hodgkin's lymphoma. Bone Marrow Transplant. 2004;**34**(10):883-890

**Chapter 10**

**Provisional chapter**

**New Horizons in Regenerative Medicine in Organ**

**New Horizons in Regenerative Medicine in Organ** 

DOI: 10.5772/intechopen.74241

Regenerative medicine is a scientific field that focuses on new approaches in the autologous repair and/or replacement of cells, tissues and/or organs. With time and technical advancements, urethral regeneration, corneal and retinal regeneration, genetically modified skin transplantation has become routine clinical and tissue reconstructive art only due to successful clinical use of stem cells and engineered tissue grafting at defined locations in respective organs in order to bring back the natural or improved physiological functions with enhanced capacity. The tissue engineering and reconstructive art are becoming integral part of the regenerative medicine. This chapter highlights the importance of regenerative medicine in successful tissue reconstruction for organ transplantation.

**Keywords:** regenerative medicine, urethral defects, bronchomalacia, limbal stem cells,

There have been exponential advances in the field of tissue engineering and regenerative medicine (TERM). There is an active and progressive focus of research to study the mechanisms of injury and how they interface and activate endogenous progenitor cell populations and with a particular focus on elucidating how progenitor cells interact with cells of the immune system. A lot of work has focused on identifying precisely which pool of stem cells

Human embryonic stem cells have an endless capacity to divide, offer an unlimited source of cells, are capable of becoming any type of cell, and can be differentiated in the laboratory.

skin transplants, amniotic stem cells, corneal and retinal stem cells

actively participates in endogenous repair/regeneration processes.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Paramjit Singh Dhot and Mayurika S. Tyagi

Paramjit Singh Dhot and Mayurika S. Tyagi

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74241

**Abstract**

**1. Introduction**

**Repair**

**Repair**


#### **New Horizons in Regenerative Medicine in Organ Repair New Horizons in Regenerative Medicine in Organ Repair**

DOI: 10.5772/intechopen.74241

Paramjit Singh Dhot and Mayurika S. Tyagi

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

Paramjit Singh Dhot and Mayurika S. Tyagi

http://dx.doi.org/10.5772/intechopen.74241

#### **Abstract**

[77] Rapoport AP, Guo C, Badros A, et al. Autologous stem cell transplantation followed by consolidation chemotherapy for relapsed or refractory Hodgkin's lymphoma. Bone

[78] Younes A, Gopal AK, Smith SE, et al. Results of a pivotal phase II study of brentuximab vedotin for patients with relapsed or refractory Hodgkin's lymphoma. J Clin Oncol.

[79] Moskowitz C, Nadamanee A, Masszi T, et al. The AETHERA trial: an ongoing phase 3 study of brentuximab vedotin in the treatment of patients at high risk of residual Hodgkin lymphom a following autologous stem cell transplant. Biol Blood and Bone

Marrow Transplant. 2004;**34**(10):883-890

2012;**30**(18):2183-2189

214 Stem Cells in Clinical Practice and Tissue Engineering

Marrow. Feb 2015;**21**(2):S28

Regenerative medicine is a scientific field that focuses on new approaches in the autologous repair and/or replacement of cells, tissues and/or organs. With time and technical advancements, urethral regeneration, corneal and retinal regeneration, genetically modified skin transplantation has become routine clinical and tissue reconstructive art only due to successful clinical use of stem cells and engineered tissue grafting at defined locations in respective organs in order to bring back the natural or improved physiological functions with enhanced capacity. The tissue engineering and reconstructive art are becoming integral part of the regenerative medicine. This chapter highlights the importance of regenerative medicine in successful tissue reconstruction for organ transplantation.

**Keywords:** regenerative medicine, urethral defects, bronchomalacia, limbal stem cells, skin transplants, amniotic stem cells, corneal and retinal stem cells

### **1. Introduction**

There have been exponential advances in the field of tissue engineering and regenerative medicine (TERM). There is an active and progressive focus of research to study the mechanisms of injury and how they interface and activate endogenous progenitor cell populations and with a particular focus on elucidating how progenitor cells interact with cells of the immune system. A lot of work has focused on identifying precisely which pool of stem cells actively participates in endogenous repair/regeneration processes.

Human embryonic stem cells have an endless capacity to divide, offer an unlimited source of cells, are capable of becoming any type of cell, and can be differentiated in the laboratory.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The use of hESC-derived cells is an attractive treatment approach, in fact, for many different diseases because transplanted cells may be able to react to changing conditions in the microenvironment, which is an important biological process.

**2.4. Tissue engineered airway**

cal disorders (**Figure 1**) [1].

3–4 CT scans).

A 30-year-old woman with end-stage bronchomalacia was transplanted with a tissue-engineered airway. Cells and MHC antigens were removed from human donor trachea and then colonised by epithelial cells, and mesenchymal stem cell-derived chondrocytes were taken from the recipient. This graft was then used to replace the recipient's left main bronchus. The graft immediately provided the recipient with a functional airway, improved her quality of life and had a normal appearance and mechanical properties at 4 months. The patient had no anti-donor antibodies and was not on immunosuppressive drugs. This case suggested that autologous cells combined with appropriate biomaterials might provide successful treatment for patient with serious clini-

New Horizons in Regenerative Medicine in Organ Repair http://dx.doi.org/10.5772/intechopen.74241 217

**Figure 1.** (A) Diagnostic CT angiography of intrahepatic portal flow (arrow), collaterals feeding the portal vein but no external portal vein in continuity (arrowhead) and enlarged spleen and collaterals around the oesophagus and in the liver hilum (open arrow). (B and C) Surgical correction showing graft attachment to the superior-mesenteric vein (arrow) and left portal vein (arrowhead). (D) Perioperative ultrasound with blood flows of 25–40 cm/s in the graft and intrahepatic portal vein. (E) Angiography showing a patent graft (arrows) 1 week after the surgery (reconstruction of

In the biomaterial area of regenerative medicine, much attention has been paid to the advancement of material design through the promotion of endogenous stem cell differentiation toward a specific phenotype of interest by modulating physical attributes of the scaffold (including stiffness, topography and porosity). Advanced materials that select refined populations and direct their differentiation may provide a mechanism to achieve improved *in vivo* regeneration.

This chapter presents the current state of the art on identifying organ defects, applying feasibility testing of regenerative host-progenitor cell interactions and finally, assessment of endogenous repair or reconstructed urethra, cornea, bronchi, retina, skin, foetal tissues with a possibility of engineered tissue transplants widely used in routine clinical practice.

### **2. Clinical success in tissue reconstruction**

#### **2.1. Urethral reconstruction for large ureteral defects**

In an interesting research area, a tissue engineering approach was used to restore function in a small group of boys with large urethral defects. Biopsies of bladder tissue were obtained, and smooth muscle and epithelial cells were isolated and cultured. The cells were then seeded onto tubular poly(glycolic acid)-poly(lactic-co-glycolic acid) scaffolds. Following culture for 1 week to ensure cell viability and matrix production, grafts were used for urethral reconstruction. The authors report that all five patients maintained functional flow rates at 36–72 months postimplantation, and biopsies confirmed that tissue organisation is similar to the native tissue.

#### **2.2. Autologous limbal stem cells in corneal damage**

Autologous stem cell therapy has been used to reverse corneal destruction due to burns. In one study, autologous limbal stem cells were cultured on fibrin to treat corneal damage in over 100 patients. At 10 years of follow-up, more than 75% of patients had a restored corneal epithelium layer. Clinical success was correlated to the percentage of functional stem cells (holoclone-forming) observed in the culture. Specifically, if cultures contained more than 3% holoclone-forming cells, clinical success was found in 78% of patients. On the other hand, if less than 3% holoclone-forming cells were found, success was seen in only 11% of the patients. This chapter elucidates the potential for limbal stem cells for corneal repair.

#### **2.3. Platelet-rich plasma in orthopaedic sports injuries**

Currently, use of platelet-rich plasma (PRP) to treat orthopaedic sports injuries is showing promise in this area. Several randomised control trials are underway, and early results have been recently published. Peerbooms *et al.* first reported the beneficial effects of PRP over corticosteroid injections for the treatment of lateral epicondylitis in a double-blind RCT. At 1 year, marked improvements were seen in both patient-reported outcomes as well as functional scores, and more recent data suggest these results persist up to 2 years.

#### **2.4. Tissue engineered airway**

The use of hESC-derived cells is an attractive treatment approach, in fact, for many different diseases because transplanted cells may be able to react to changing conditions in the micro-

In the biomaterial area of regenerative medicine, much attention has been paid to the advancement of material design through the promotion of endogenous stem cell differentiation toward a specific phenotype of interest by modulating physical attributes of the scaffold (including stiffness, topography and porosity). Advanced materials that select refined populations and direct their differentiation may provide a mechanism to achieve improved

This chapter presents the current state of the art on identifying organ defects, applying feasibility testing of regenerative host-progenitor cell interactions and finally, assessment of endogenous repair or reconstructed urethra, cornea, bronchi, retina, skin, foetal tissues with a possibility of engineered tissue transplants widely used in routine clinical practice.

In an interesting research area, a tissue engineering approach was used to restore function in a small group of boys with large urethral defects. Biopsies of bladder tissue were obtained, and smooth muscle and epithelial cells were isolated and cultured. The cells were then seeded onto tubular poly(glycolic acid)-poly(lactic-co-glycolic acid) scaffolds. Following culture for 1 week to ensure cell viability and matrix production, grafts were used for urethral reconstruction. The authors report that all five patients maintained functional flow rates at 36–72 months postimplantation, and biopsies confirmed that tissue organisation is similar to the native tissue.

Autologous stem cell therapy has been used to reverse corneal destruction due to burns. In one study, autologous limbal stem cells were cultured on fibrin to treat corneal damage in over 100 patients. At 10 years of follow-up, more than 75% of patients had a restored corneal epithelium layer. Clinical success was correlated to the percentage of functional stem cells (holoclone-forming) observed in the culture. Specifically, if cultures contained more than 3% holoclone-forming cells, clinical success was found in 78% of patients. On the other hand, if less than 3% holoclone-forming cells were found, success was seen in only 11% of the patients.

Currently, use of platelet-rich plasma (PRP) to treat orthopaedic sports injuries is showing promise in this area. Several randomised control trials are underway, and early results have been recently published. Peerbooms *et al.* first reported the beneficial effects of PRP over corticosteroid injections for the treatment of lateral epicondylitis in a double-blind RCT. At 1 year, marked improvements were seen in both patient-reported outcomes as well as functional scores, and more recent data suggest these results persist up to 2 years.

This chapter elucidates the potential for limbal stem cells for corneal repair.

environment, which is an important biological process.

216 Stem Cells in Clinical Practice and Tissue Engineering

**2. Clinical success in tissue reconstruction**

**2.1. Urethral reconstruction for large ureteral defects**

**2.2. Autologous limbal stem cells in corneal damage**

**2.3. Platelet-rich plasma in orthopaedic sports injuries**

*in vivo* regeneration.

A 30-year-old woman with end-stage bronchomalacia was transplanted with a tissue-engineered airway. Cells and MHC antigens were removed from human donor trachea and then colonised by epithelial cells, and mesenchymal stem cell-derived chondrocytes were taken from the recipient. This graft was then used to replace the recipient's left main bronchus. The graft immediately provided the recipient with a functional airway, improved her quality of life and had a normal appearance and mechanical properties at 4 months. The patient had no anti-donor antibodies and was not on immunosuppressive drugs. This case suggested that autologous cells combined with appropriate biomaterials might provide successful treatment for patient with serious clinical disorders (**Figure 1**) [1].

**Figure 1.** (A) Diagnostic CT angiography of intrahepatic portal flow (arrow), collaterals feeding the portal vein but no external portal vein in continuity (arrowhead) and enlarged spleen and collaterals around the oesophagus and in the liver hilum (open arrow). (B and C) Surgical correction showing graft attachment to the superior-mesenteric vein (arrow) and left portal vein (arrowhead). (D) Perioperative ultrasound with blood flows of 25–40 cm/s in the graft and intrahepatic portal vein. (E) Angiography showing a patent graft (arrows) 1 week after the surgery (reconstruction of 3–4 CT scans).

#### **2.5. Transplant of genetically modified skin**

Recently, Dr Michele De Luca, MD and his colleagues in Italy saved the life of a boy who had lost most of his epidermis by life-saving regeneration of virtually the entire epidermis. Patient's own epidermal stem cells were genetically modified to have functional copies of the gene that was mutant [2].

The boy was presented with blistered skin, which is the characteristic of junctional epidermolysis bullosa (JEB), and associated bacterial skin infections. Within days, about 60% of his epidermis had vanished. LAMB3 is one of three genes that encode a laminin protein that links the epidermis to the dermis.

The researchers cultured primary keratinocytes from a 4-cm2 biopsy specimen from an unblistered area in the boy's left inguinal region. Then, they used retroviral vectors to introduce LAMB3 genes. The grafts grew. The genetic modification of those cells by introducing extra copies of the LAMB3 gene restored the epidermal machinery.

Three types of cultures grew into grafts from the boy's cells: holoclones, which are all stem cells; paraclones, which are specialised cells and meroclones, which are partly differentiated cells. The transgenic grafts harbour all three types of clones, but only the holoclones persist.

Procedures were done to cover the affected areas with genetically modified and regenerated grafts. The patches were up to several inches in diameter and were applied on a properly prepared wound bed. After engraftment, the epidermis looks basically normal, and that is also true at the molecular level in terms of the adhesion machinery that has been replaced. Within 5 weeks, the cells had covered about 80% of the boy's body. Even hairs grew, which usually does not happen with the conventional skin grafts. This case suggested that transgenic epidermal stem cells can regenerate a fully functional epidermis virtually indistinguishable from a normal epidermis, in the absence of related adverse events [2].

#### **2.6. Amniotic-derived tissue grafts for enhanced skin regeneration**

Amniotic tissues contain many regenerative cytokines, growth factors and extracellular matrix molecules, including proteoglycans, hyaluronic acid and collagens I, III and IV. Dehydrated amnion/chorion grafts are currently used to treat a variety of wounds such as diabetic foot ulcers and burns. In a recent study, Mowry et al. [3] found that all amniotic-derived tissue grafts appeared to stimulate improved healing over sham wounds (ungrafted wounds), evidenced by more normal appearing dermal matrix architecture, epidermal structure and maturity. In addition, the hypothermically stored amniotic membrane (HSAM) grafts promoted greater tissue regeneration than the dehydrated amnion/chorion (dHACM) meshed grafts, as measured by the presence of basket-weave collagen matrix and formation of follicles and glands (**Figures 2** and **3**).

immunomodulation of the wound environment [4]. This study highlights the importance of

**Figure 2.** Schematic of the composition of amniotic-derived products. (A) Basic structure of native placental tissues outlining amniotic membrane, which interfaces with the foetus and major components such as epithelium, basement membrane, stromal layer and the spongy layer and the chorion which interfaces maternal tissues. Schematic of

New Horizons in Regenerative Medicine in Organ Repair http://dx.doi.org/10.5772/intechopen.74241 219

Retinal pigment epithelial (RPE) cells derived from human embryonic stem cells can be safely transplanted into the eyes of the patients with retinal degeneration, with early signs of vision

Two teams of researchers (Dr Eyal Banin from Israel and Dr. Ninel Z. Gregori from Florida) reported preliminary findings from phase 1 and phase 2 trials at the American Academy of Ophthalmology (AAO) 2017 Annual Meeting [5]. Patients had the dry form of age-related macular degeneration(AMD) or Stargardt disease and received injections of human embryonic stem cell (hESC)-derived RPE cells. Results of the studies are optimistic even though the

processing techniques and how they influence the quality of wound healing.

(B) dehydrated amnion/chorion and (C) hypothermically stored amniotic membrane.

**2.7. Stem cell therapy in retinal degeneration**

gain, according to pioneers in the field.

Current studies point to several critical factors that may contribute to enhanced wound repair with amniotic-derived tissues including ECM, cytokines and growth factors, stem cells and

**Figure 2.** Schematic of the composition of amniotic-derived products. (A) Basic structure of native placental tissues outlining amniotic membrane, which interfaces with the foetus and major components such as epithelium, basement membrane, stromal layer and the spongy layer and the chorion which interfaces maternal tissues. Schematic of (B) dehydrated amnion/chorion and (C) hypothermically stored amniotic membrane.

immunomodulation of the wound environment [4]. This study highlights the importance of processing techniques and how they influence the quality of wound healing.

#### **2.7. Stem cell therapy in retinal degeneration**

**2.5. Transplant of genetically modified skin**

218 Stem Cells in Clinical Practice and Tissue Engineering

The researchers cultured primary keratinocytes from a 4-cm2

copies of the LAMB3 gene restored the epidermal machinery.

a normal epidermis, in the absence of related adverse events [2].

**2.6. Amniotic-derived tissue grafts for enhanced skin regeneration**

gene that was mutant [2].

the epidermis to the dermis.

persist.

Recently, Dr Michele De Luca, MD and his colleagues in Italy saved the life of a boy who had lost most of his epidermis by life-saving regeneration of virtually the entire epidermis. Patient's own epidermal stem cells were genetically modified to have functional copies of the

The boy was presented with blistered skin, which is the characteristic of junctional epidermolysis bullosa (JEB), and associated bacterial skin infections. Within days, about 60% of his epidermis had vanished. LAMB3 is one of three genes that encode a laminin protein that links

tered area in the boy's left inguinal region. Then, they used retroviral vectors to introduce LAMB3 genes. The grafts grew. The genetic modification of those cells by introducing extra

Three types of cultures grew into grafts from the boy's cells: holoclones, which are all stem cells; paraclones, which are specialised cells and meroclones, which are partly differentiated cells. The transgenic grafts harbour all three types of clones, but only the holoclones

Procedures were done to cover the affected areas with genetically modified and regenerated grafts. The patches were up to several inches in diameter and were applied on a properly prepared wound bed. After engraftment, the epidermis looks basically normal, and that is also true at the molecular level in terms of the adhesion machinery that has been replaced. Within 5 weeks, the cells had covered about 80% of the boy's body. Even hairs grew, which usually does not happen with the conventional skin grafts. This case suggested that transgenic epidermal stem cells can regenerate a fully functional epidermis virtually indistinguishable from

Amniotic tissues contain many regenerative cytokines, growth factors and extracellular matrix molecules, including proteoglycans, hyaluronic acid and collagens I, III and IV. Dehydrated amnion/chorion grafts are currently used to treat a variety of wounds such as diabetic foot ulcers and burns. In a recent study, Mowry et al. [3] found that all amniotic-derived tissue grafts appeared to stimulate improved healing over sham wounds (ungrafted wounds), evidenced by more normal appearing dermal matrix architecture, epidermal structure and maturity. In addition, the hypothermically stored amniotic membrane (HSAM) grafts promoted greater tissue regeneration than the dehydrated amnion/chorion (dHACM) meshed grafts, as measured by the presence of basket-weave collagen matrix and formation of follicles and glands (**Figures 2** and **3**). Current studies point to several critical factors that may contribute to enhanced wound repair with amniotic-derived tissues including ECM, cytokines and growth factors, stem cells and

biopsy specimen from an unblis-

Retinal pigment epithelial (RPE) cells derived from human embryonic stem cells can be safely transplanted into the eyes of the patients with retinal degeneration, with early signs of vision gain, according to pioneers in the field.

Two teams of researchers (Dr Eyal Banin from Israel and Dr. Ninel Z. Gregori from Florida) reported preliminary findings from phase 1 and phase 2 trials at the American Academy of Ophthalmology (AAO) 2017 Annual Meeting [5]. Patients had the dry form of age-related macular degeneration(AMD) or Stargardt disease and received injections of human embryonic stem cell (hESC)-derived RPE cells. Results of the studies are optimistic even though the

**Acknowledgements**

**Conflict of interest**

**Author details**

**References**

Authors have no conflict of interest.

2008;**372**(9655):2023-2030

2017;**29**(9):277

Paramjit Singh Dhot\* and Mayurika S. Tyagi

\*Address all correspondence to: psd2682@gmail.com Saraswathi Institute of Medical Sciences, Hapur, India

Repair and Regeneration. 2014;**22**(4):451-456

[1] Macchiarini P et al. Clinical transplantation of a tissue-engineered airway. The Lancet.

New Horizons in Regenerative Medicine in Organ Repair http://dx.doi.org/10.5772/intechopen.74241 221

[3] Mowry KC, Bonvallet PP, Bellis SL. Enhanced skin regeneration using a novel amniotic-derived tissue graft. Wounds: A Compendium of Clinical Research and Practice.

[4] Litwiniuk M, Grzela T. Amniotic membrane: New concepts for an old dressing. Wound

[5] Stem Cell Therapy Shows Promise for Retinal Degeneration. Medscape. Nov 16, 2017

[2] Stem Cell Gene Therapy Replaces Boy's Entire Epidermis. Medscape. Nov 8, 2017

Authors acknowledge knowledge database for this study.

**Figure 3.** Twenty-one day representative histological sections: (A) sham; (B) dehydrated amnion-chorion (dHACM) meshed; (C) dHACM; and (D) hypothermically stored amniotic membrane (HSAM). Histologic summary: Highmagnification images were used to assess the wounds qualitatively, and representative images are shown in this figure. Amniotic-derived grafts stimulated more robust healing and wound repair than sham wounds. Interestingly, HSAMtreated wounds displayed early epidermal formation, reconstitution of dermal appendages and a high degree of the basket-weave matrix, thus producing regenerated skin tissue that closely mimics unwounded skin at 21 days.

studies are very early and very small. This is just a first step in the long road toward making regenerative cell therapy a reality in macular and retinal degeneration.

Dysfunction and degeneration of RPE cells contribute to vision loss in AMD. In both studies, human embryonic stem cells were turned into RPE cells and injected into the sub-retinal space of the patients with retinal degeneration at a dose of 50,000–200,000 cells. The expectation is that, once in place, the new RPE cells will support or replace the patient's own failing RPE cells and boost the survival of photoreceptors.

### **3. Conclusion**

With science and technical advancement in the regenerative medicine and tissue engineering, recent research data suggest detailed investigative studies on the mechanisms of endogenous injury, interactions at organ or tissue cell interface with activated endogenous progenitor cell populations, with a particular focus on mechanisms of how progenitor cells behave with cells of the immune system. Several reports suggest success in identifying pool of stem cells transplanted and actively participated in endogenous corneal, retinal, epicondyle, skin, bronchi and foetal organ repair/regeneration processes.

### **Acknowledgements**

Authors acknowledge knowledge database for this study.

### **Conflict of interest**

Authors have no conflict of interest.

### **Author details**

Paramjit Singh Dhot\* and Mayurika S. Tyagi

\*Address all correspondence to: psd2682@gmail.com

Saraswathi Institute of Medical Sciences, Hapur, India

### **References**

studies are very early and very small. This is just a first step in the long road toward making

**Figure 3.** Twenty-one day representative histological sections: (A) sham; (B) dehydrated amnion-chorion (dHACM) meshed; (C) dHACM; and (D) hypothermically stored amniotic membrane (HSAM). Histologic summary: Highmagnification images were used to assess the wounds qualitatively, and representative images are shown in this figure. Amniotic-derived grafts stimulated more robust healing and wound repair than sham wounds. Interestingly, HSAMtreated wounds displayed early epidermal formation, reconstitution of dermal appendages and a high degree of the basket-weave matrix, thus producing regenerated skin tissue that closely mimics unwounded skin at 21 days.

Dysfunction and degeneration of RPE cells contribute to vision loss in AMD. In both studies, human embryonic stem cells were turned into RPE cells and injected into the sub-retinal space of the patients with retinal degeneration at a dose of 50,000–200,000 cells. The expectation is that, once in place, the new RPE cells will support or replace the patient's own failing RPE

With science and technical advancement in the regenerative medicine and tissue engineering, recent research data suggest detailed investigative studies on the mechanisms of endogenous injury, interactions at organ or tissue cell interface with activated endogenous progenitor cell populations, with a particular focus on mechanisms of how progenitor cells behave with cells of the immune system. Several reports suggest success in identifying pool of stem cells transplanted and actively participated in endogenous corneal, retinal, epicondyle, skin, bronchi

regenerative cell therapy a reality in macular and retinal degeneration.

cells and boost the survival of photoreceptors.

220 Stem Cells in Clinical Practice and Tissue Engineering

and foetal organ repair/regeneration processes.

**3. Conclusion**


**Section 4**

**Tissue Engineering Mechanisms and Stem Cell**

**Based Product Manufacturing**

**Tissue Engineering Mechanisms and Stem Cell Based Product Manufacturing**

**Chapter 11**

**Provisional chapter**

**Optimal Delivery Strategy for Stem Cell Therapy in**

Stem cell therapy is a new strategy for patients with ischemic heart disease. However, no consensus exists on the most optimal delivery strategy, but an important factor that determines the success of stem cell therapy is the choice of cell delivery route to the heart. Delivery strategy affects the fate of cells and subsequently influences outcome of proce‐ dure. Our review summarizes current approaches for administration of stem cells to the heart. Three most used approaches are intracoronary, intramyocardial, and epicardial injection. They have been widely used for delivery of different types of cells. There are several advantages of these stem cell administration approaches, but stem cell retention and stem cell survival rates are quite low using these methods, which might limit their therapeutic effects. Alternative attempts to improve current stem cell therapy methods are reviewed along with emerging new stem cell delivery approaches. The present chap‐ ter displays the current status on stem cell delivery techniques, their efficacy, and clinical

**Keywords:** stem cell therapy, delivery method, ischemic heart disease, intramyocardial

**Optimal Delivery Strategy for Stem Cell Therapy in** 

DOI: 10.5772/intechopen.69537

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

Regenerative medicine with stem cell therapy has been tested in clinical trials in patients with ischemic heart disease [1]. The aim of this method is to induce growth of new blood vessels in the myocardium or replacement of damaged myocardial cells either directly by differentia‐

tion of stem cells or by a paracrine effect of cytokines secreted from the stem cells.

**Patients with Ischemic Heart Disease**

**Patients with Ischemic Heart Disease**

Andrei Cismaru and Gabriel Cismaru

Andrei Cismaru and Gabriel Cismaru

http://dx.doi.org/10.5772/intechopen.69537

success in different trials.

injection

**1. Introduction**

**Abstract**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

### **Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease Patients with Ischemic Heart Disease**

**Optimal Delivery Strategy for Stem Cell Therapy in** 

DOI: 10.5772/intechopen.69537

Andrei Cismaru and Gabriel Cismaru Andrei Cismaru and Gabriel Cismaru Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69537

#### **Abstract**

Stem cell therapy is a new strategy for patients with ischemic heart disease. However, no consensus exists on the most optimal delivery strategy, but an important factor that determines the success of stem cell therapy is the choice of cell delivery route to the heart. Delivery strategy affects the fate of cells and subsequently influences outcome of proce‐ dure. Our review summarizes current approaches for administration of stem cells to the heart. Three most used approaches are intracoronary, intramyocardial, and epicardial injection. They have been widely used for delivery of different types of cells. There are several advantages of these stem cell administration approaches, but stem cell retention and stem cell survival rates are quite low using these methods, which might limit their therapeutic effects. Alternative attempts to improve current stem cell therapy methods are reviewed along with emerging new stem cell delivery approaches. The present chap‐ ter displays the current status on stem cell delivery techniques, their efficacy, and clinical success in different trials.

**Keywords:** stem cell therapy, delivery method, ischemic heart disease, intramyocardial injection

#### **1. Introduction**

Regenerative medicine with stem cell therapy has been tested in clinical trials in patients with ischemic heart disease [1]. The aim of this method is to induce growth of new blood vessels in the myocardium or replacement of damaged myocardial cells either directly by differentia‐ tion of stem cells or by a paracrine effect of cytokines secreted from the stem cells.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

When applied to the heart stem cell therapy, it has several important factors that might influ‐ ence therapeutic success, including the properties of stem cells and type of the disease that affect the heart of host.

reserved for patients necessitating coronary artery bypass grafting (CABG) with direct tho‐ racotomy. Catheter‐based intramyocardial delivery is limited by the technology of catheters and mapping systems. In patients with recent myocardial ischemia and injury due to a signifi‐ cant stenosis or occlusion of a coronary artery, intracoronary artery delivery may not be the optimal route regardless of experimental results with the technique. In this case, intravenous

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

227

Each of the techniques has its own advantages and disadvantages. The optimal method is still unclear. Studies have used either intravenous, intracoronary, or intramyocardial injection.

In 2001, the first rodent study with stem cells was published by Orlic et al., showing improve‐ ment in the heart function by regeneration [4]. Six months later, the first clinical trial on humans reported positive results for intracoronary injection of bone marrow stem cells after

Starting with the preclinical pioneering work of Orlic et al., intramyocardial and intravenous deliveries of BMC have been shown to improve left ventricular function in ischemic heart

• The transcoronary catheter‐based procedure was first performed by Thompson et al. using a catheter in combination with an intravascular ultrasound imaging and demonstrated in swines that are feasible and safe [6]. Few years later, Siminiak et al. finished the first phase I clinical trial with his method confirming the feasibility and safety of the procedure in

• The transendocardial technique was first used in a swine model by Fuchs et al. who dem‐ onstrated improvement of the cardiac function [8]. Since then, clinical studies have been

• The first trial of bone marrow stem cells in chronic ischemic cardiomyopathy was per‐ formed by Perin et al. [9]. He studied percutaneous transendocardial injection of stem cells

• For the intravenous infusion, the safety and feasibility have been confirmed using a swine

• In a swine model of myocardial injury, Vicario et al. [12] and Yokoyama et al. [13] demon‐ strated that retrograde coronary sinus injection does not produce significant hemodynamic changes and reported presence of autologous bone marrow stem cells in the myocardium.

The systemic route of delivery is simple, not so invasive, but retention to the heart of stem cells is very low. Higher rates of retention were seen with mesenchymal cells because of their homing capacity. This route needs to be associated with methods of enhancing homing to the

model [10] as well as later in a phase I clinical study on humans [11].

or intracoronary venous injection is preferred.

acute myocardial infarction [5].

published with positive results.

and provided encouraging results.

*2.1.1. Intravenous delivery*

disease (**Table 1**).

humans [7].

**2.1. Chronology of optimal delivery developments**

The choice of the delivery methods is also very important because this will affect the reten‐ tion rate, survival, integration in the host, and functionality of stem cells. Therefore, delivery method influences the subsequent outcome of this new emerging treatment [2].

The aim of this review is to discuss methods of delivery in regenerative stem cell therapy in patients with ischemic heart disease. We will focus on current issues derived from conducted clinical trials and emerging new approaches.

### **2. Routes to the heart: advantages and disadvantages**

Different approaches for delivering cells to the heart were developed and are utilized in pre‐ clinical and clinical current studies: intramyocardial (IM), intracoronary (IC), and intravenous (IV) (**Figure 1**) approaches were widely used, but no method currently meets the criteria of a perfect delivery method [3]. A stepwise approach of optimal delivery would consider if the patient needs open chest surgery. Surgical intramyocardial delivery is the most direct but

**Figure 1.** Major routes for delivering stem cells to the heart.

reserved for patients necessitating coronary artery bypass grafting (CABG) with direct tho‐ racotomy. Catheter‐based intramyocardial delivery is limited by the technology of catheters and mapping systems. In patients with recent myocardial ischemia and injury due to a signifi‐ cant stenosis or occlusion of a coronary artery, intracoronary artery delivery may not be the optimal route regardless of experimental results with the technique. In this case, intravenous or intracoronary venous injection is preferred.

Each of the techniques has its own advantages and disadvantages. The optimal method is still unclear. Studies have used either intravenous, intracoronary, or intramyocardial injection.

#### **2.1. Chronology of optimal delivery developments**

When applied to the heart stem cell therapy, it has several important factors that might influ‐ ence therapeutic success, including the properties of stem cells and type of the disease that

The choice of the delivery methods is also very important because this will affect the reten‐ tion rate, survival, integration in the host, and functionality of stem cells. Therefore, delivery

The aim of this review is to discuss methods of delivery in regenerative stem cell therapy in patients with ischemic heart disease. We will focus on current issues derived from conducted

Different approaches for delivering cells to the heart were developed and are utilized in pre‐ clinical and clinical current studies: intramyocardial (IM), intracoronary (IC), and intravenous (IV) (**Figure 1**) approaches were widely used, but no method currently meets the criteria of a perfect delivery method [3]. A stepwise approach of optimal delivery would consider if the patient needs open chest surgery. Surgical intramyocardial delivery is the most direct but

method influences the subsequent outcome of this new emerging treatment [2].

**2. Routes to the heart: advantages and disadvantages**

affect the heart of host.

226 Stem Cells in Clinical Practice and Tissue Engineering

clinical trials and emerging new approaches.

**Figure 1.** Major routes for delivering stem cells to the heart.

In 2001, the first rodent study with stem cells was published by Orlic et al., showing improve‐ ment in the heart function by regeneration [4]. Six months later, the first clinical trial on humans reported positive results for intracoronary injection of bone marrow stem cells after acute myocardial infarction [5].

Starting with the preclinical pioneering work of Orlic et al., intramyocardial and intravenous deliveries of BMC have been shown to improve left ventricular function in ischemic heart disease (**Table 1**).


#### *2.1.1. Intravenous delivery*

The systemic route of delivery is simple, not so invasive, but retention to the heart of stem cells is very low. Higher rates of retention were seen with mesenchymal cells because of their homing capacity. This route needs to be associated with methods of enhancing homing to the


**Clinical trial Administration Reference** 2011 Williams et al. i.m. [69] 2011 Perin et al. i.m. [70] 2011 Povsic et al. i.m. [71] 2011 Duckers et al. i.m.. [72] 2011 Hirsch et al. HEBE Intracoronary [73] 2011 Roncali et al. BONAMI Intracoronary [74] 2011 Traverse et al. Late TIME Intracoronary [75] 2011 Quyyumi Intracoronary [76] 2011 Tuma Retrograde coronary [45] 2011 Moreira Retrograde coronary [46] 2012 Makkar et al. CADUCEUS Intracoronary [77] 2013 Bolli et al. SCIPIO Intracoronary [78] 2013 Vrtovec Intracoronary [79] 2013 Huang Intracoronary [80] 2013 Kurbonov et al. Intracoronary [81] 2013 Forcillo et al Via CABG + i.m. [82] 2014 Assmann et al. Via CABG+epicardial [83] 2014 Nasseri et al i.m. [84] 2014 Brickwedel et al. Via CABG [85]

2014 Hong Intracoronary + retrograde coronary sinus

i.m., intramyocardial.

2015 Hao Intracoronary [87] 2015 Chang Intracoronary [88] 2015 Gao Intracoronary [89] 2015 Fiarresga Intracoronary [90] 2015 Helseth Intracoronary [91] 2015 Eirin Intrarenal [92] 2015 Lee Intracoronary [93] 2016 Tseliou Intracoronary [94] 2017 Xiao Intracoronary [95]

**Table 1.** Cell delivery in studies and publications on myocardial infarction and chronic ischemic heart failure.

[86]

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

229


**Clinical trial Administration Reference** 2002 TOPCARE‐AMI Intracoronary [14] 2004 Perin et al. Intracoronary [47] 2004 BOOST Intracoronary [16] 2004 Chen et al. Intracoronary [38] 2004 Siminiak et al. Epicardial [48] 2005 Katritsis et al. Intracoronary [36] 2005 Erbs et al. Intracoronary [49] 2006 REPAIR‐AMI Intracoronary [50] 2006 Assmus et al. Intracoronary [51] 2006 ASTAMI Intracoronary [19] 2006 Chen et al. Intracoronary [39] 2006 Fuchs et al. Intracoronary [34, 35] 2006 Beeres et al. Intracoronary [52] 2006 Hendrikx Epicardial [53] 2006 Kang et al. Intracoronary [54] 2007 Losordo et al. i.m. [30] 2007 Katritsis et al. Intracoronary [36] 2007 Mohyeddin‐Bonab et al. i.m. [55] 2007 Beeres et al. Intracoronary [56] 2007 Ahmadi et al. i.m. [57] 2008 Diederichsen et al. Intracoronary [58] 2008 FINCELL Intracoronary [59] 2008 Menasche et al. Epicardial [60] 2008 HEBE Intracoronary [61] 2008 Beeres et al. Intracoronary [62] 2009 Hare et al. i.v. [11] 2009 Van Ramshorst et al. i.m. [63] 2009 BALANCE Intracoronary [64] 2009 MYSTAR Intracoronary/i.m. [65] 2009 REGENT Intracoronary [66]

228 Stem Cells in Clinical Practice and Tissue Engineering

2010 Kastrup et al. i.m.

2010 Strauer et al. Intracoronary [67] 2011 Yerebakan et al. Epicardial [68]

**Table 1.** Cell delivery in studies and publications on myocardial infarction and chronic ischemic heart failure.

ischemic tissue because most of the stem cells show localization in other tissues with only a small part of injected cells engrafted at the level of the heart [14–17]. This method may be limited to acute myocardial infarction and not be suitable for chronic ischemic heart disease because it relies on physiologic homing signals present few days after an acute myocardial infarction.

#### *2.1.2. Intracoronary delivery*

An attractive method is intracoronary infusion because it can disseminate relatively uni‐ formly cells to the entire region infused [18]. It is also widely available, less invasive than intramyocardial method, and it is used in numerous clinical trials [14–22]. Intracoronary infu‐ sion implies a percutaneous approach typically through the femoral artery with a standard balloon catheter. The catheter used for delivery infuses cells to the myocardial regions in which blood supply is preserved. For injection, balloon occlusion is needed in order to reduce the washout into the systemic circulation and increase adhesion of cells and transmigration of the infused cells to the myocardium [19–24].

One reason to use this method is the familiarity between interventional cardiologists. The method is less invasive than injection directly in the myocardium and requires the equipment standardly found in a catheterization laboratory. The method enables a relatively homoge‐ neous dissemination of stem cells to the target area.

The disadvantage of this method is that some adult stem cells such as autologous cardio‐ sphere‐derived cells [25] or mesenchymal stem cells (MSCs) [26, 27], produced microvascular occlusion after intracoronary delivery, raising concerns over the use of this method delivery in patients with ischemic heart disease. Actually, the diameter of autologous cardiosphere‐ derived cells and mesenchymal stem cells is around 20 μm, which could exceed the diameter of some arterioles [28]. The great majority of clinical studies use this approach to inject smaller cells such as bone marrow mononuclear cells. Another disadvantage could be the poor reten‐ tion rate following intracoronary injection. This is caused by the loss of a high proportion of stem cells in the systemic circulation during several minutes.

In a comparative study [26], intramyocardial technique was compared to intravenous delivery and intracoronary method. Intramyocardial injection of MSCs was better than intracoronarian delivery in terms of blood flow to the myocardium. In dogs and also in pigs micro‐infarctions were seen probably due to cell micro‐thrombi which created obstruction to the blood flow when injected through intracoronary path. In humans, this complication was not seen [36–39]. One disadvantage of intramyocardial injection is the formation of islet‐like clusters of stem cells at this level. *Another disadvantage of intramyocardial injection is association with a higher risk* 

**Figure 2.** Intramyocardial injection at the border zone between healthy myocardium and dense necrosis. The border

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

231

Epicardial injection is performed using a needle‐syringe system under direct visualization of the operated heart. This method is the most used in preclinical research using animal models. The intramyocardial delivery of stem cells can be achieved by direct injection after open thora‐ cotomy (sternotomy or left thoracotomy). Most of the time this method is used in conjunction

*of ventricular arrhythmias than with other methods of deliver* [26].

*2.1.4. Intramyocardial‐transepicardial injection*

zone is probably hibernating but viable myocardium.

#### *2.1.3. Intramyocardial‐transendocardial injection*

Transendocardial injection is performed percutaneously and is less invasive than epicardial injection [29, 30] but more invasive than intracoronary injection. The access is made by punc‐ ture of the femoral artery or vein (transseptal approach) and then the catheter is passed in the left ventricle. An electroanatomical map of the left ventricle is realized in order to navigate inside the cavity and position the injection catheter to specific areas (**Figure 2**).

Electroanatomical mapping system permits left ventricular mapping and guides injection to the border zone between healthy and necrosed endocardium [26, 31–35]. Intramyocardial injection of bone marrow‐derived stem cells and also angiogenic genes has been reported to be safe in terms of arrhythmia or death [26, 30–35]. On the other hand, intramyocardial injec‐ tion of skeletal myoblasts has been shown to have a pro‐arrhythmogenic effect [26, 31–35].

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease http://dx.doi.org/10.5772/intechopen.69537 231

In a comparative study [26], intramyocardial technique was compared to intravenous delivery and intracoronary method. Intramyocardial injection of MSCs was better than intracoronarian delivery in terms of blood flow to the myocardium. In dogs and also in pigs micro‐infarctions were seen probably due to cell micro‐thrombi which created obstruction to the blood flow when injected through intracoronary path. In humans, this complication was not seen [36–39].

One disadvantage of intramyocardial injection is the formation of islet‐like clusters of stem cells at this level. *Another disadvantage of intramyocardial injection is association with a higher risk of ventricular arrhythmias than with other methods of deliver* [26].

#### *2.1.4. Intramyocardial‐transepicardial injection*

ischemic tissue because most of the stem cells show localization in other tissues with only a small part of injected cells engrafted at the level of the heart [14–17]. This method may be limited to acute myocardial infarction and not be suitable for chronic ischemic heart disease because it relies on physiologic homing signals present few days after an acute myocardial

An attractive method is intracoronary infusion because it can disseminate relatively uni‐ formly cells to the entire region infused [18]. It is also widely available, less invasive than intramyocardial method, and it is used in numerous clinical trials [14–22]. Intracoronary infu‐ sion implies a percutaneous approach typically through the femoral artery with a standard balloon catheter. The catheter used for delivery infuses cells to the myocardial regions in which blood supply is preserved. For injection, balloon occlusion is needed in order to reduce the washout into the systemic circulation and increase adhesion of cells and transmigration of

One reason to use this method is the familiarity between interventional cardiologists. The method is less invasive than injection directly in the myocardium and requires the equipment standardly found in a catheterization laboratory. The method enables a relatively homoge‐

The disadvantage of this method is that some adult stem cells such as autologous cardio‐ sphere‐derived cells [25] or mesenchymal stem cells (MSCs) [26, 27], produced microvascular occlusion after intracoronary delivery, raising concerns over the use of this method delivery in patients with ischemic heart disease. Actually, the diameter of autologous cardiosphere‐ derived cells and mesenchymal stem cells is around 20 μm, which could exceed the diameter of some arterioles [28]. The great majority of clinical studies use this approach to inject smaller cells such as bone marrow mononuclear cells. Another disadvantage could be the poor reten‐ tion rate following intracoronary injection. This is caused by the loss of a high proportion of

Transendocardial injection is performed percutaneously and is less invasive than epicardial injection [29, 30] but more invasive than intracoronary injection. The access is made by punc‐ ture of the femoral artery or vein (transseptal approach) and then the catheter is passed in the left ventricle. An electroanatomical map of the left ventricle is realized in order to navigate

Electroanatomical mapping system permits left ventricular mapping and guides injection to the border zone between healthy and necrosed endocardium [26, 31–35]. Intramyocardial injection of bone marrow‐derived stem cells and also angiogenic genes has been reported to be safe in terms of arrhythmia or death [26, 30–35]. On the other hand, intramyocardial injec‐ tion of skeletal myoblasts has been shown to have a pro‐arrhythmogenic effect [26, 31–35].

inside the cavity and position the injection catheter to specific areas (**Figure 2**).

infarction.

*2.1.2. Intracoronary delivery*

230 Stem Cells in Clinical Practice and Tissue Engineering

the infused cells to the myocardium [19–24].

neous dissemination of stem cells to the target area.

stem cells in the systemic circulation during several minutes.

*2.1.3. Intramyocardial‐transendocardial injection*

Epicardial injection is performed using a needle‐syringe system under direct visualization of the operated heart. This method is the most used in preclinical research using animal models.

The intramyocardial delivery of stem cells can be achieved by direct injection after open thora‐ cotomy (sternotomy or left thoracotomy). Most of the time this method is used in conjunction with cardiac surgery such as coronary artery bypass grafting (CABG) or left ventricular assist device (LVAD) [40−42]. Myocardial retention rates have been similar to those of a transendocardial approach [1, 3, 43]. The advantage is that in some types of necrosis: intra‐ myocardial, epicardial, or combined, targeted tissue can be reached only through a direct epicardial access.

By this, a left ventricular endocardial map is obtained with electromechanical information that characterizes the underlying tissue and permits to navigate into the heart. This real map helps to precisely localize the injection catheter at the level of necrosis, border zone of necrosis or healthy tissue. The map is constructed by acquiring multiple points at different locations in the left ventricle from base to apex, from inferior to anterior, and from septal to lateral. These anatomical points with electrical value are gated to a surface electrocardio‐ gram. Ultra‐low magnetic fields are generated by the system using a triangular magnetic pad placed under the patient and other three patches positioned on the thorax of the patient. Each point sample contains electrical information about local activity such as unipolar volt‐ age and local contractility such as linear local shortening. The resulting tridimensional map of the left ventricle gives information about electromechanical properties of the myocar‐ dium and is able to distinguish between ischemic areas (which have low lineal local short‐ ening and preserved unipolar voltage) from infarcted areas (low linear local shortening and

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

233

Most of the studies using NOGA tridimensional system for intramyocardial delivery used the conventional right femoral approach in order to reach the endocardium. However there are cases with tortuous, angled right iliac artery, it implies difficulty in advancing the mapping catheter to the left ventricle and manipulating it inside the cavity. When the right femoral artery cannot be used, then left artery can be tried, and in case of failure, other arteries (like

The NOGA mapping system was designed for the transfemoral approach. This precludes its use in patients who have peripheral vascular disease with intense calcified and tortuous iliac or femoral arteries. Because NOGA catheters are advanced into the LV without using a guidewire, manipulation can be difficult inside the heart especially when arteries are tortu‐ ous and do not permit free rotation and angulation of the catheter. Manipulation can be even more challenging when using a stiffer injection catheter. Although there has been no formal recommendation concerning alternative approaches in patients with peripheral arterial tor‐ tuosity, there are reports showing a benefit of using radial artery or femoral vein and trans‐ septal approach in this category of patients. In the case of tortuous iliac or femoral arteries, the

To date, there are still many unanswered questions regarding delivery methods in stem cell therapy. Some of these questions will be answered in the ongoing trials. Larger double‐ blinded placebo‐controlled clinical trials are needed to elucidate whether it is trans‐aortic or transseptal approach. It is the best method to reach different zones of endocardial necrosis. In

radial) or veins (femoral vein with transseptal approach) can be accessed.

brachial access could be taken in order to avoid procedural complications.

low unipolar voltage) [1].

**3.1. Transfemoral approach with the NOGA system**

**3.2. Transradial approach with the NOGA system**

**4. Perspectives for 2017–2020**

Other options not so invasive like open chest thoracotomy have been tried: minimally inva‐ sive lateral thoracotomy using thoracoscopic injection to the epicardium and minimally inva‐ sive subxifoidian technique using robotic devices [44].

#### *2.1.5. Intracoronary sinus injection*

Another technique to inject stem cells to the heart of animals or humans [45−47] is through the coronary sinus or tributary veins. The percutaneous approach is made through the femoral vein. With the use of a catheter that passes in the right atrium, one can cannulate the coronary sinus and access the middle cardiac vein, the great cardiac vein, or other tributaries of the coronary sinus. For injection, balloon occlusion is needed in order to reduce the washout into the systemic circulation. Comparing with the intracoronary injection, this method has the advantage of lower risk of coronary embolism and injection can be made even in areas with low arterial supply.

### **3. Electroanatomical mapping using the NOGA system**

Intramyocardial injection is the method by which stem cell suspension is directly injected in the myocardium using a needle. This method needs electroanatomical mapping in order to identify the zone of necrosis. Intramyocardial injection enables stem cells to be targeted into this localized area. In patients with new or old myocardial infarction, stem cells are usually injected at the border zone of the infarct with the healthy tissue. This area has a relatively good blood supply to ensure stem cell survival compared to the infarcted area with no blood flow. Intramyocardial injection permits to target zones even with low blood flow. Intracoronary injection instead requires a normal flow through a coronary artery. Intramyocardial injection enables cells to be delivered to areas with a limited vascularity. Because this method has no risk of coronary embolism, larger cells can be used, like skeletal myoblasts, mesenchymal stem cells, and others.

The current system for intramyocardial delivery is the NOGA® XP Cardiac Navigation System (Biologics Delivery Systems Group of Cordis Corporation, a Johnson & Johnson Company). This system is able to perform electromechanical mapping of both left ventricle and right ventricle. Electromechanical mapping permits clear delineation of the targeted area and precise deployment of the therapeutic product [3]. This delivery method has proved to be feasible in the presence of chronic ischemic heart disease and acute myocar‐ dial infarction (within 10 days after infarction). The system incorporates an injection cath‐ eter and the real‐time reconstruction of the heart's endocardial surface in three dimensions using collection of points with spatial, electrophysiologic, and mechanical information. By this, a left ventricular endocardial map is obtained with electromechanical information that characterizes the underlying tissue and permits to navigate into the heart. This real map helps to precisely localize the injection catheter at the level of necrosis, border zone of necrosis or healthy tissue. The map is constructed by acquiring multiple points at different locations in the left ventricle from base to apex, from inferior to anterior, and from septal to lateral. These anatomical points with electrical value are gated to a surface electrocardio‐ gram. Ultra‐low magnetic fields are generated by the system using a triangular magnetic pad placed under the patient and other three patches positioned on the thorax of the patient. Each point sample contains electrical information about local activity such as unipolar volt‐ age and local contractility such as linear local shortening. The resulting tridimensional map of the left ventricle gives information about electromechanical properties of the myocar‐ dium and is able to distinguish between ischemic areas (which have low lineal local short‐ ening and preserved unipolar voltage) from infarcted areas (low linear local shortening and low unipolar voltage) [1].

#### **3.1. Transfemoral approach with the NOGA system**

with cardiac surgery such as coronary artery bypass grafting (CABG) or left ventricular assist device (LVAD) [40−42]. Myocardial retention rates have been similar to those of a transendocardial approach [1, 3, 43]. The advantage is that in some types of necrosis: intra‐ myocardial, epicardial, or combined, targeted tissue can be reached only through a direct

Other options not so invasive like open chest thoracotomy have been tried: minimally inva‐ sive lateral thoracotomy using thoracoscopic injection to the epicardium and minimally inva‐

Another technique to inject stem cells to the heart of animals or humans [45−47] is through the coronary sinus or tributary veins. The percutaneous approach is made through the femoral vein. With the use of a catheter that passes in the right atrium, one can cannulate the coronary sinus and access the middle cardiac vein, the great cardiac vein, or other tributaries of the coronary sinus. For injection, balloon occlusion is needed in order to reduce the washout into the systemic circulation. Comparing with the intracoronary injection, this method has the advantage of lower risk of coronary embolism and injection can be made even in areas with

Intramyocardial injection is the method by which stem cell suspension is directly injected in the myocardium using a needle. This method needs electroanatomical mapping in order to identify the zone of necrosis. Intramyocardial injection enables stem cells to be targeted into this localized area. In patients with new or old myocardial infarction, stem cells are usually injected at the border zone of the infarct with the healthy tissue. This area has a relatively good blood supply to ensure stem cell survival compared to the infarcted area with no blood flow. Intramyocardial injection permits to target zones even with low blood flow. Intracoronary injection instead requires a normal flow through a coronary artery. Intramyocardial injection enables cells to be delivered to areas with a limited vascularity. Because this method has no risk of coronary embolism, larger cells can be used, like skeletal myoblasts, mesenchymal

The current system for intramyocardial delivery is the NOGA® XP Cardiac Navigation System (Biologics Delivery Systems Group of Cordis Corporation, a Johnson & Johnson Company). This system is able to perform electromechanical mapping of both left ventricle and right ventricle. Electromechanical mapping permits clear delineation of the targeted area and precise deployment of the therapeutic product [3]. This delivery method has proved to be feasible in the presence of chronic ischemic heart disease and acute myocar‐ dial infarction (within 10 days after infarction). The system incorporates an injection cath‐ eter and the real‐time reconstruction of the heart's endocardial surface in three dimensions using collection of points with spatial, electrophysiologic, and mechanical information.

epicardial access.

low arterial supply.

stem cells, and others.

*2.1.5. Intracoronary sinus injection*

232 Stem Cells in Clinical Practice and Tissue Engineering

sive subxifoidian technique using robotic devices [44].

**3. Electroanatomical mapping using the NOGA system**

Most of the studies using NOGA tridimensional system for intramyocardial delivery used the conventional right femoral approach in order to reach the endocardium. However there are cases with tortuous, angled right iliac artery, it implies difficulty in advancing the mapping catheter to the left ventricle and manipulating it inside the cavity. When the right femoral artery cannot be used, then left artery can be tried, and in case of failure, other arteries (like radial) or veins (femoral vein with transseptal approach) can be accessed.

#### **3.2. Transradial approach with the NOGA system**

The NOGA mapping system was designed for the transfemoral approach. This precludes its use in patients who have peripheral vascular disease with intense calcified and tortuous iliac or femoral arteries. Because NOGA catheters are advanced into the LV without using a guidewire, manipulation can be difficult inside the heart especially when arteries are tortu‐ ous and do not permit free rotation and angulation of the catheter. Manipulation can be even more challenging when using a stiffer injection catheter. Although there has been no formal recommendation concerning alternative approaches in patients with peripheral arterial tor‐ tuosity, there are reports showing a benefit of using radial artery or femoral vein and trans‐ septal approach in this category of patients. In the case of tortuous iliac or femoral arteries, the brachial access could be taken in order to avoid procedural complications.

### **4. Perspectives for 2017–2020**

To date, there are still many unanswered questions regarding delivery methods in stem cell therapy. Some of these questions will be answered in the ongoing trials. Larger double‐ blinded placebo‐controlled clinical trials are needed to elucidate whether it is trans‐aortic or transseptal approach. It is the best method to reach different zones of endocardial necrosis. In cases of intramyocardial or epicardial necrosis, epicardial approach should be compared with endocardial one. Brachial can be an option for patients who have peripheral vascular disease with impossible femoral approach. Novel biomedical *engineering* is used in several emerging technologies for delivering stem cells to the heart. These include transplantation of stem cells as tissue‐engineered constructs [80]. All these delivery options will permit a more individual and personalized stem cell treatment strategy in patients with ischemic heart disease.

[4] Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocar‐

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

235

[5] Strauer BE, Brehm M, Zeus T, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation.

[6] Thompson CA, Nasseri BA, Makower J, et al. Percutaneous transvenous cellular cardio‐ myoplasty: A novel nonsurgical approach for myocardial cell transplantation. Journal of

[7] Siminiak T, Fiszer D, Jerzykowska O, et al. Percutaneous trans‐coronary‐venous transplantation of autologous skeletal myoblasts in the treatment of post‐infarction myocardial contractility impairment: The POZNAN trial. European Heart Journal.

[8] Fuchs S, Baffour R, Zhou YF, Shou M, Pierre A, Tio FO, Weissman NJ, Leon MB, Epstein SE, Kornowski R. Transendocardial delivery of autologous bone marrow enhances col‐ lateral perfusion and regional function in pigs with chronic experimental myocardial

[9] Perin EC, Dohmann HF, Borojevic R, et al. Transendocardial, autologous bone mar‐ row cell transplantation for severe, chronic ischemic heart failure. Circulation. 2003;

[10] Halkos ME, Zhao ZQ, Kerendi F, et al. Intravenous infusion of mesenchymal stem cells enhances regional perfusion and improves ventricular function in a porcine model of

[11] Hare JM, Traverse JH, Henry TD, et al. A randomized, double‐blind, placebo‐controlled, dose‐escalation study of intravenous adult human mesenchymal stem cells (prochy‐ mal) after acute myocardial infarction. Journal of the American College of Cardiology.

[12] Vicario J, Piva J, Pierini A, et al. Transcoronary sinus delivery of autologous bone mar‐ row and angiogenesis in pig models with myocardial injury. Cardiovascular Radiation

[13] Yokoyama SI, Fukuda N, Li Y, et al. A strategy of retrograde injection of bone mar‐ row mononuclear cells into the myocardium for the treatment of ischemic heart disease.

[14] Assmus B, Schächinger V, Teupe C, Britten M, Lehmann R, Döbert N, Grünwald F, Aicher A, Urbich C, Martin H, Hoelzer D, Dimmeler S, Zeiher AM. Transplantation of pro‐ genitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE‐

[15] Fernández‐Avilés F, San Román JA, García‐Frade J, Fernández ME, Peñarrubia MJ, de la Fuente L, Gómez‐Bueno M, Cantalapiedra A, Fernández J, Gutierrez O, Sánchez PL,

Journal of Molecular and Cellular Cardiology. 2006;**40**(1):24‐34

myocardial infarction. Basic Research in Cardiology. 2008;**103**(6):525‐536

ischemia. Journal of the American College of Cardiology. 2001:**37**(6):1726‐1732

the American College of Cardiology. 2003;**41**(11):1964‐1971

dium. Nature. 2001;**410**:701‐705

2002;**106**:1913‐1918

2005;**26**(12):1188‐1195

**107**:2294‐2302

2009;**54**(24):2277‐2286

Medicine. 2002;**3**(2):91‐94

AMI). Circulation. 2002;**106**:3009‐3017

### **5. Conclusions**

There are several methods of cell delivery to the heart. However, none of these are perfect for every type of ischemic disease or every stem cell type. Advantages and disadvantages of each technique will help in tailoring the treatment protocol for every individual patient and will aid in planning future clinical trials. Combining these techniques (e.g. intracoronary artery + intracoronary sinus injection) could reduce washout and increase adhesion to the necrosed area. Emerging new approaches need to be also developed for the future of clinical success using stem cell therapy administered for ischemic heart disease.

### **Author details**

Andrei Cismaru<sup>1</sup> and Gabriel Cismaru<sup>2</sup> \*

\*Address all correspondence to: gabi\_cismaru@yahoo.com

1 11th Department of Oncology, Medical Oncology and Radiotherapy, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj‐Napoca, Romania

2 5th Department of Internal Medicine, Cardiology‐Rehabilitation, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj‐Napoca, Romania

### **References**


[4] Orlic D, Kajstura J, Chimenti S, et al. Bone marrow cells regenerate infarcted myocar‐ dium. Nature. 2001;**410**:701‐705

cases of intramyocardial or epicardial necrosis, epicardial approach should be compared with endocardial one. Brachial can be an option for patients who have peripheral vascular disease with impossible femoral approach. Novel biomedical *engineering* is used in several emerging technologies for delivering stem cells to the heart. These include transplantation of stem cells as tissue‐engineered constructs [80]. All these delivery options will permit a more individual

There are several methods of cell delivery to the heart. However, none of these are perfect for every type of ischemic disease or every stem cell type. Advantages and disadvantages of each technique will help in tailoring the treatment protocol for every individual patient and will aid in planning future clinical trials. Combining these techniques (e.g. intracoronary artery + intracoronary sinus injection) could reduce washout and increase adhesion to the necrosed area. Emerging new approaches need to be also developed for the future of clinical success

1 11th Department of Oncology, Medical Oncology and Radiotherapy, Iuliu Hatieganu

2 5th Department of Internal Medicine, Cardiology‐Rehabilitation, Iuliu Hatieganu University

[1] Perin EC, Lopez J. Methods of stem cell delivery in cardiac diseases. Nature Clinical

[2] Perin EC, Silva GV, Assad JA, Vela D, Buja LM, Sousa AL, Litovsky S, Lin J, Vaughn WK, Coulter S, Fernandes MR, Willerson JT. Comparison of intracoronary and transendo‐ cardial delivery of allogeneic mesenchymal cells in a canine model of acute myocardial

[3] Psaltis PJ, Zannettino AC, Gronthos S, Worthley SG. Intramyocardial navigation and mapping for stem cell delivery. Journal of Cardiovascular Translational Research.

infarction. Journal of Molecular and Cellular Cardiology. 2008;**44**:486‐495

and personalized stem cell treatment strategy in patients with ischemic heart disease.

using stem cell therapy administered for ischemic heart disease.

\*

and Gabriel Cismaru<sup>2</sup>

of Medicine and Pharmacy, Cluj‐Napoca, Romania

\*Address all correspondence to: gabi\_cismaru@yahoo.com

University of Medicine and Pharmacy, Cluj‐Napoca, Romania

Practice Cardiovascular Medicine. 2006;**3**(Suppl 1):S110‐113

**5. Conclusions**

234 Stem Cells in Clinical Practice and Tissue Engineering

**Author details**

Andrei Cismaru<sup>1</sup>

**References**

2010;**3**(2):135‐146


Hernández C, Sanz R, García‐ Sancho J, Sánchez A. Experimental and clinical regen‐ erative capability of human bone marrow cells after myocardial infarction. Circulation Research. 2004;**95**:742‐748

benefits of autologous cardiosphere‐derived cells in porcine ischemic cardiomyopathy.

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

237

[26] Freyman T, Polin G, Osman H, Crary J, Lu M, Cheng L, Palasis M, Wilensky RL. A quan‐ titative, randomized study evaluating three methods of mesenchymal stem cell delivery

[27] Vulliet PR, Greeley M, Halloran SM, MacDonald KA, Kittleson MD. Intra‐coronary arterial injection of mesenchymal stromal cells and microinfarction in dogs. Lancet.

[28] Kassab GS, Fung YC. Topology and dimensions of pig coronary capillary network.

[29] Krause K, Jaquet K, Schneider C, Haupt S, Lioznov MV, Otte KM, Kuck KH. Percutaneous intramyocardial stem cell injection in patients with acute myocardial in farction: First‐

[30] Losordo DW, Schatz RA, White CJ, Udelson JE, Veereshwarayya V, Durgin M, et al. Intramyocardial transplantation of autologous CD34+ stem cells for intractable angin a.

[31] Fernandes S, Amirault JC, Lande G, et al. Autologous myoblast transplantation after myo‐ cardial infarction increases the inducibility of ventricular arrhythmias. Cardiovascular

[32] Smits PC, van Geuns RJ, Poldermans D, et al. Catheter‐based intramyocardial injection of autologous skeletal myoblasts as a primary treatment of ischemic heart failure: Clinical experience with six‐month follow‐up. Journal of the American College of Cardiology.

[33] Veltman CE, Soliman OI, Geleijnse ML, et al. Four‐year followup of treatment with intra‐ myocardial skeletal myoblasts injection in patients with ischaemic cardiomyopathy.

[34] Fuchs S, Kornowski R, Weisz G, et al. Safety and feasibility of transendocardial autol‐ ogous bone marrow cell transplantation in patients with advanced heart disease.

[35] Baldazzi F, Jorgensen E, Ripa RS, et al. Release of biomarkers of myocardial damage after direct intramyocardial injection of genes and stem cells via the percutaneous trans‐

[36] Katritsis DG, Sotiropoulou PA, Karvouni E, et al. Transcoronary transplantation of autologous mesenchymal stem cells and endothelial progenitors into infarcted human

myocardium. Catheterization and Cardiovascular Interventions. 2005;**65**:321‐329 [37] Katritsis DG, Sotiropoulou P, Giazitzoglou E, et al. Electrophysiological effects of intra‐ coronary transplantation of autologous mesenchymal and endothelial progenitor cells.

following myocardial infarction. European Heart Journal. 2006;**27**:1114‐1122

American Journal of Physiology. 1994;**267**(1 pt 2):H319‐H325

inman study. Heart. 2009;**95**(14):1145‐1152

European Heart Journal. 2008;**29**:1386‐1396

American Journal of Cardiology. 2006;**97**:823‐829

luminal route. European Heart Journal. 2008;**29**:1819‐1826

Circ ulation. 2007;**115**(25):3165‐3172

Research. 2006;**69**:348‐358

Europace. 2007;**9**:167‐171

2003;**42**:2063‐2069

Circulation. 2009;**120**:1075‐1083

2004;**363**:783‐784


benefits of autologous cardiosphere‐derived cells in porcine ischemic cardiomyopathy. Circulation. 2009;**120**:1075‐1083

[26] Freyman T, Polin G, Osman H, Crary J, Lu M, Cheng L, Palasis M, Wilensky RL. A quan‐ titative, randomized study evaluating three methods of mesenchymal stem cell delivery following myocardial infarction. European Heart Journal. 2006;**27**:1114‐1122

Hernández C, Sanz R, García‐ Sancho J, Sánchez A. Experimental and clinical regen‐ erative capability of human bone marrow cells after myocardial infarction. Circulation

[16] Wollert KC, Meyer GP, Lotz J, Ringes‐Lichtenberg S, Lippolt P, Breidenbach C, Fichtner S, Korte T, Hornig B, Messinger D, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary autologous bone marrow cell transfer after myocardial infarction: the

[17] Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, Kalantzi M, Herbots L, Sinnaeve P, Dens J, Maertens J, Rademakers F, Dymarkowski S, Gheysens O, Van Cleemput J, Bormans G, Nuyts J, Belmans A, Mortelmans L, Boogaerts M, Van de Werf F. Autologous bone marrow‐derived stem‐cell transfer in patients with ST‐segment elevation myocardial infarction: Double‐blind, randomised controlled trial. Lancet.

[18] Li Q, Guo Y, Ou Q, Chen N, Wu WJ, Yuan F, O'Brien E, Wang T, Luo L, Hunt GN, Zhu X, Bolli R. Intracoronary administration of cardiac stem cells in mice: A new, improved tech‐ nique for cell therapy in murine models. Basic Research in Cardiology. 2011;**106**:849‐864

[19] Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, Endresen K, Ilebekk A, Mangschau A, Fjeld JG, Smith HJ, Taraldsrud E, Grøgaard HK, Bjørnerheim R, Brekke M, Müller C, Hopp E, Ragnarsson A, Brinchmann JE, Forfang K. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. The New England

[20] Erbs S, Linke A, Schuler G, Hambrecht R. Intracoronary administration of circulating blood‐derived progenitor cells after recanalization of chronic coronary artery occlusion

[21] Abdel‐Latif A, Bolli R, Tleyjeh IM, Montori VM, Perin EC, Hornung CA, Zuba‐Surma EK, Al‐Mallah M, Dawn B. Adult bone marrow‐derived cells for cardiac repair: A sys‐ tematic review and meta‐analysis. Archives of Internal Medicine. 2007;**167**:989‐997 [22] Meyer GP, Wollert KC, Lotz J, Steffens J, Lippolt P, Fichtner S, Hecker H, Schaefer A, Arseniev L, Hertenstein B, Ganser A, Drexler H. Intracoronary bone marrow cell trans‐ fer after myocardial infarction: Eighteen months' follow‐up data from the randomized, controlled BOOST (BOne marrow transfer to enhance ST‐elevation infarct regeneration)

[23] Schachinger V, Erbs S, Elsasser A, et al. Intracoronary bone marrow‐derived pro‐ genitor cells in acute myocardial infarction. The New England Journal of Medicine.

[24] Kastrup J. Gene therapy and angiogenesis in patients with coronary artery disease.

[25] Johnston PV, Sasano T, Mills K, Evers R, Lee ST, Smith RR, Lardo AC, Lai S, Steenbergen C, Gerstenblith G, Lange R, Marbán E. Engraftment, differentiation, and functional

improves endothelial function. Circulation Research. 2006;**98**:e48

Expert Review of Cardiovascular Therapy. 2010;**8**(8):1127‐1138

BOOST randomized controlled clinical trial. Lancet. 2004;**364**:141‐148

Research. 2004;**95**:742‐748

236 Stem Cells in Clinical Practice and Tissue Engineering

2006;**367**:113‐121

Journal of Medicine. 2006;**355**:1199‐1209

trial. Circulation. 2006;**113**:1287‐1294

2006;**355**:1210‐1221


[38] Chen SL, Fang WW, Ye F, et al. Effect on left ventricular function of intracoronary trans‐ plantation of autologous bone marrow mesenchymal stem cell in patients with acute myocardial infarction. American Journal of Cardiology. 2004;**94**:92‐95

[50] Schachinger V, Erbs S, Elsasser A, et al. Improved clinical outcome after intracoronary administration of bone‐marrow‐derived progenitor cells in acute myocardial infarc‐ tion: Final 1‐year results of the REPAIR‐AMI trial. European Heart Journal. 2006;**27**(23):

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

239

[51] Assmus B, Honold B, Schächinger V, et al. Transcoronary transplantation of pro‐ genitor cells after myocardial infarction. The New England Journal of Medicine.

[52] Beeres SLMA, Bax JJ, Kaandorp TAM, et al. Usefulness of intramyocardial injection of autologous bone marrow‐derived mononuclear cells in patients with severe angina pectoris and stressinduced myocardial ischemia. American Journal of Cardiology.

[53] Hendrikx M, Hensen K, Clijsters C, et al. Recovery of regional but not global contractile function by the direct intramyocardial autologous bone marrow transplantation: Results from a randomized controlled clinical trial. Circulation. 2006;**114**(1Suppl):I101‐107

[54] Kang HJ, Lee HY, Na SH, et al. Differential effect of intracoronary infusion of mobilized peripheral blood stem cells by granulocyte colony–stimulating factor on left ventricular function and remodeling in patients with acute myocardial infarction versus old myo‐ cardial infarction. The MAGIC Cell‐3‐DES randomized, controlled trial. Circulation.

[55] Mohyeddin‐Bonab M, Mohamad‐Hassani MR, Alimoghaddam K, et al. Autologous in vitro expanded mesenchymal stem cell therapy for human old myocardial infarction.

[56] Beeres SLMA, Bax JJ, Dibbets‐Schneider P, et al. Intramyocardial injection of autolo‐ gous bone marrow mononuclear cells in patients with chronic myocardial infarction and severe leftventricular dysfunction. American Journal of Cardiology. 2007;**100**:1094‐1098

[57] Ahmadi H, Baharvand H, Ashtiani SK, et al. Safety analysis and improved cardiac func‐ tion following local autologous transplantation of CD133(+) enriched bone marrow cells

[58] Diederichsen ACP, Møller JE, Thayssen P, et al. Effect of intracoronary injection of bene marrow cells in patients with ischaemic heart failure. The Danish Stem Cell study— Congestive Heart Failure trial (DanCell‐CHF). European Journal of Heart Failure.

[59] Huikuri HV, Kervinen K, Niemela M, et al. Effects of intracoronary injection of mono‐ nuclear bone marrow cells on left ventricular function, arrhythmia risk profile, and restenosis after thrombolytic therapy of acute myocardial infarction. European Heart

[60] Menasche P, Alfieri O, Janssens S, et al. The myoblastAutologous grafting in ischemic cardiomyopathy (MAGIC) trial: First randomized placebo‐controlled study of myoblast

after myocardial infarction. Current Neurovascular Research. 2007;**4**(3):153‐160

2775‐2783

2006;**355**:1222‐1232

2006;**97**:1326‐1331

2006;**114**:I‐145‐I‐151

2008;**10**:661‐667

Journal. 2008;**29**:2723‐2732

Archives of Iranian Medicine. 2007;**10**(4):467‐473

transplantation. Circulation. 2008;**117**(9):1189‐1200


[50] Schachinger V, Erbs S, Elsasser A, et al. Improved clinical outcome after intracoronary administration of bone‐marrow‐derived progenitor cells in acute myocardial infarc‐ tion: Final 1‐year results of the REPAIR‐AMI trial. European Heart Journal. 2006;**27**(23): 2775‐2783

[38] Chen SL, Fang WW, Ye F, et al. Effect on left ventricular function of intracoronary trans‐ plantation of autologous bone marrow mesenchymal stem cell in patients with acute

[39] Chen S, Liu Z, Tian N, et al. Intracoronary transplantation of autologous bone marrow mesenchymal stem cells for ischemic cardiomyopathy due to isolated chronic occluded

[40] Zhao Q, Sun Y, Xia L, Chen A, Wang Z. Ran‐ domized study of mononuclear bone marrow cell transplantation in patients with coronary surgery. The Annals of Thoracic

[41] Dib N, Michler RE, Pagani FD, Wright S, Kereiakes DJ, Lengerich R, et al. Safety and feasibility of autolo‐ gous myoblast transplantation in patients with ischemic cardio‐

[42] Ota T, Patronik NA, Schwartzman D, Riviere CN, Zenati MA. Minimally invasive epi‐ cardial injections using a novel semiautonomous robotic device. Circulation. 2008;**118**(14

[43] Dib N, Dinsmore J, Lababidi Z, White B, Moravec S, Campbell A, et al. One‐year follow‐ up of feasibility and safety of the first U.S., randomized, controlled study using 3‐dimen‐ sional guided catheter‐based delivery of autologous skeletal myoblasts for ischemic cardiomyopathy (CAuSMIC study). JACC Cardiovascular Interventions. 2009;**2**(1):9‐16

[44] Arom KV, Ruengsakulrach P, Jotisakulratana V. Intramyocardial angiogenic cell pre‐ cursor injection for cardiomyopathy. Asian Cardiovascular and Thoracic Annals.

[45] Tuma J, Fernanadez‐Vina R, Carrasco A, Castillo J, Cruz C, Carrillo A, et al. Safety and feasibility of percutane‐ ous retrograde coronary sinus delivery of autologous bone mar‐ row mononuclear cell transplantation in patients with chronic refractory angina. Journal

[46] Moreira Rde C, Haddad AF, Silva SA, Souza AL, Tuche FA, Oliveira MA, et al. Intracoronary stem‐cell injection after myocardial infarction: Microcirculation sub‐

[47] Perin EC, Dohmann HF, Borojevic R, et al. Improved exercise capacity and ischemia 6 and 12 months after transendocardial injection of autologous bone marrow mono‐

[48] Siminiak T, Kalawski R, Fiszer D, Jerzykowska O, Rzezniczak J, Rozwadowska N, et al. Autologous skeletal myoblast transplantation for the treatment of postinfarction myocardial injury: Phase I clinical study with 12 months of follow‐up. American Heart

[49] Erbs S, Linke A, Adams V, et al. Transplantation of blood‐derived progenitor cells after recanalization of chronic coronary artery occlusion: First randomized and placebo‐con‐

nuclear cells for ischemic cardiomyopathy. Circulation. 2004;**110**:II213‐218

study. Arquivos Brasileiros de Cardiologia. 2011;**97**(5):420‐426

trolled study. Circulation Research. 2005;**97**(8):756‐762

left anterior descending artery. Journal of Invasive Cardiology. 2006;**8**:552‐556

myocardial infarction. American Journal of Cardiology. 2004;**94**:92‐95

Surgery. 2008;**86**(6):1833‐1840

238 Stem Cells in Clinical Practice and Tissue Engineering

suppl 1):S115‐S120

2008;**16**(2):143‐148

myopathy. Circulation. 2005;**112**(12):1748‐1755

of Translational Medicine. 2011;**9**(1):183

Journal. 2004;**148**(3):531‐537


[61] Hirsch A, Nijveldt R, van der Vleuten PA, et al. Intracoronary infusion of autologous mononuclear bone marrow cells in patients with acute myocardial infarction treated with primary PCI: Pilot study of the multicenter HEBE trial. Catheterization and Cardiovascular Interventions. 2008;**71**:273‐281

effects of skeletal myoblast implantation by catheter deliv‐ ery in patients with chronic heart failure after myocardial infarction. American Heart Journal. 2011;**162**(4):654.

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

241

[72] Duckers HJ, Houtgraaf J, Hehrlein C, Schofer J, Waltenberger J, Gershlick A, et al. Final results of a phase IIa, rando‐ mised, open‐label trial to evaluate the percutaneous intra‐ myocardial transplantation of autologous skeletal myoblasts in congestive heart failure

[73] Hirsch A, Nijveldt R, van der Vleuten PA, Tijssen JGP, van der Giessen WJ, Tio RA, et al. Intracoronary infusion of mononuclear cells from bone marrow or peripheral blood compared with standard therapy in patients after acute myo‐ cardial infarction treated by primary percutaneous coronary inter‐ vention: Results of the randomized controlled

[74] Roncalli J, Mouquet F, Piot C, Trochu J‐N, Le Corvoisier P, Neuder Y, et al. Intracoronary autologous mononu‐ cleated bone marrow cell infusion for acute myocardial infarc‐ tion: Results of the randomized multicenter BONAMI trial. European Heart Journal.

[75] Traverse JH, Henry TD, Ellis SG, Pepine CJ, Willerson JT, Zhao DXM, et al. Effect of intracoronary delivery of autologous bone marrow mononuclear cells 2 to 3 weeks fol‐ lowing acute myocardial infarction on left ventricular function. JAMA: The Journal of

[76] Quyyumi AA, Waller EK, Murrow J, Esteves F, Galt J, Oshinski J, et al. CD34(+) cell infu‐ sion after ST elevation myocardial infarction is associated with improved perfusion and

[77] Makkar RR, Smith RR, Cheng K, Malliaras K, Thomson LEJ, Berman D, et al. Intracoronary cardiosphere‐ derived cells for heart regeneration after myocardial infarction (CADUCEUS): a prospective, randomised phase 1 trial. The Lancet. 2012;**379**(9819):895‐904

[78] Bolli R, Chugh AR, D'Amario D, Loughran JH, Stoddard MF, Ikram S, et al. Cardiac stem cells in patients with ischaemic cardiomyopathy (SCIPIO): Initial results of a ran‐

[79] Chiu LL, Iyer RK, Reis LA, Nunes SS, Radisic M. Cardiac tissue engineering: Current state and perspectives. Frontiers in Bioscience: A Journal and Virtual Library. 2012;**17**:

[80] Zhao X, Huang L. Cardiac stem cells. A promising treatment option for heart failure.

[81] Kurbonov U, Dustov A, Barotov A, et al. Intracoronary infision of autologous CD133 cells in myocardial infarction and tracing TC99MIBI scintigraphy of the heart areas involved in cell homing. Stem Cells International. 2013;**2013**:ID 582527. http://dx.doi.

patients: The SEISMIC trial. EuroIntervention. 2011;**6**(7):805‐812

HEBE trial. European Heart Journal. 2011;**32**(14):1736‐1747

the American Medical Association. 2011;**306**(19):2110‐2119

is dose dependent. American Heart Journal. 2011;**161**(1):98‐105

domised phase 1 trial. The Lancet. 2011;**378**(9806):1847‐1857

Experimental and Therapeutic Medicine. 2013;**5**:379‐383

e1‐662.e1

2011;**32**(14):1748‐1757

1533‐1535

org/10.1155/2013/582527


effects of skeletal myoblast implantation by catheter deliv‐ ery in patients with chronic heart failure after myocardial infarction. American Heart Journal. 2011;**162**(4):654. e1‐662.e1

[72] Duckers HJ, Houtgraaf J, Hehrlein C, Schofer J, Waltenberger J, Gershlick A, et al. Final results of a phase IIa, rando‐ mised, open‐label trial to evaluate the percutaneous intra‐ myocardial transplantation of autologous skeletal myoblasts in congestive heart failure patients: The SEISMIC trial. EuroIntervention. 2011;**6**(7):805‐812

[61] Hirsch A, Nijveldt R, van der Vleuten PA, et al. Intracoronary infusion of autologous mononuclear bone marrow cells in patients with acute myocardial infarction treated with primary PCI: Pilot study of the multicenter HEBE trial. Catheterization and

[62] Beeres SLMA, Lamb HJ, Roes SD, et al. Effect of intramyocardial bone marrow cell injection on diastolic function in patients with chronic myocardial isc hemia. Journal of

[63] Van Ramshorst J, Bax JJ, Beeres SLMA, et al. Intramyocardial bone marrow cell injec‐ tion for chronic myocardial ischemia: A randomized controlled trial. The Journal of the

[64] Yousef M, Schannwell CM, Kostering M, et al. The BALANCE study: Clinical benefit and long‐term outcome after intracoronary autologous bone marrow cell transplanta‐ tion in patients with acute myocardial infarction. Journal of the American College of

[65] Gyongyosi M, Lang I, Dettke M, et al. Combined delivery approach of bone marrow mononuclear stem cells early and late after myocardial infarction: The MYSTAR prospec‐ tive, randomized study. Nature Clinical Practice Cardiovascular Medicine. 2009;**6**:70‐81

[66] Tendera M, Wojakowski W, Ruzyłło W, et al. Intracoronary infusion of bone marrow‐ derived selected CD34+CXCR4+ cells and non‐selected mononuclear cells in patients with acute STEMI and reduced left ventricular ejection fraction: Results of randomized, multicentre Myocardial Regeneration by Intracoronary Infusion of Selected Population of Stem Cells in Acute Myocardial Infarction (REGENT) Trial. European Heart Journal.

[67] Strauer BE, Yousef M, Schannwell CM. The acute and long‐term effects of intracoronary Stem cell Transplantation in 191 patients with chronic HeSRt failure: The STAR‐heart

[68] Yerebakan C, Kaminski A, Westphal B, Donndorf P, Glass A, Liebold A, et al. Impact of preoperative left ventric‐ ular function and time from infarction on the long‐term benefits after intramyocardial CD133+ bone marrow stem cell transplant. The Journal of

[69] Williams AR, Trachtenberg B, Velazquez DL, McNiece I, Altman P, Rouy D, et al. Intramyocardial stem cell injection in patients with ischemic cardiomyopathy/novelty

[70] Perin EC, Silva GV, Henry TD, Cabreira‐Hansen MG, Moore WH, Coulter SA, et al. A randomized study of transendocardial injection of autologous bone marrow mono‐ nuclear cells and cell function analysis in ischemic heart. American Heart Journal.

[71] Povsic TJ, O'ConnorCM, Henry T, Taussig A, Kereiakes DJ, Fortuin FD, et al. A double‐ blind, randomized, controlled, multicenter study to assess the safety and cardiovas‐ cular

Cardiovascular Interventions. 2008;**71**:273‐281

Magnetic Resonance Imaging. 2008;**27**:992‐997

Cardiology. 2009;**53**:2262‐2269

240 Stem Cells in Clinical Practice and Tissue Engineering

2009;**30**:1313‐1321

2011;**161**(6):1078‐1087

American Medical Association. 2009;**301**(19):1997‐2004

study. European Journal of Heart Failure. 2010;**12**:721‐729

Thoracic and Cardiovascular Surgery. 2011;**142**(6):1530.e3‐1539.e3

and s i gnifi cance. Circulation Research. 2011;**108**(7):792‐796


[82] Forcillo J, Stevens L‐M, Mansour S, et al. Implantation of CD133+stem cells in patients undergoing coronary bypass surgery: IMPACT‐CABG pilot trial. Canadian Journal of Cardiology. 2013;**29**:441‐447. ID 582527. http://dx.doi.org/10.1155/2013/582527

[93] Lee HW, Lee HC, Park JH, et al. Effects of intracoronary administration of autologous asipose tissue‐derived stem cells on acute myocardial infarction in porcine model.

Optimal Delivery Strategy for Stem Cell Therapy in Patients with Ischemic Heart Disease

http://dx.doi.org/10.5772/intechopen.69537

243

[94] Tseliou E, Kanazawa H, Dawkins J, et al. Widespread myocardial delivery of heart‐ derived stem cells by nonoclusive triple‐vessel intracoronary infusion in porcine isch‐ emic cardiomyopathy: Superior attenuation of adverse remodeling documented by

[95] Xiao W, Guo S, Gao C, Dai G, Gao Y, et al. A randomized comparative studyon the efficacy of intracoronary infusion of autologous bone marrow mononuclear cells and mesenchymal stem cells in patients with dilated cardiomyopathy. International Heart

magnetc reasonnce imaging and histology. PLoS ONE. 2016;**11**:e0144523

Journal. Int Heart J 2017;**58**:238‐244. doi: 10.1536/ihj.16‐328

Yonsei Medical Journal. 2015;**56**:1522‐1529


[93] Lee HW, Lee HC, Park JH, et al. Effects of intracoronary administration of autologous asipose tissue‐derived stem cells on acute myocardial infarction in porcine model. Yonsei Medical Journal. 2015;**56**:1522‐1529

[82] Forcillo J, Stevens L‐M, Mansour S, et al. Implantation of CD133+stem cells in patients undergoing coronary bypass surgery: IMPACT‐CABG pilot trial. Canadian Journal of

[83] Assmann A, Heke M, Kropil P, et al. Laser‐supported CD133+ cell therapy in patients with ischemic cardiomyopathy: Initial results from a prospective phase I multicenter

[84] Nasseri BA, Ebell W, Dandel M, et al. Autologous CD133+bone marrow cells and bypass grafting for regeneration of ischaemic myocardium: The Cardio133 trial. European

[85] Brickwedel J, Gulbins H, Reichenspurner H. Long‐term follow‐up after autologous skel‐ etal myoblast transplantation in ischaemic heart disease. Interactive Cardiovascular and

[86] Hong SJ, Hou D, Brinton TJ, Johnstone B, Feng D, et al. Intracoronary and retrograde coronary venous myocardial delivery of adipose‐derived stem cells in swine infarc‐ tion lead to transient myocardil trapping with predominant pulmonary distribution.

[87] Hao L, Hao J, Fang W, Han C, Zhang W, Wang X. Dual isotope simultaneous imaging to evaluate the effects of intracoronary bone marrow derived mesenchymal stem cells on perfusion and metabolism in canines with acute myocardial infarction. Biomedical

[88] Chung ES, Miller L, Patel AN, et al. Changes in ventricular remodeling and clinical sta‐ tus during the year following a single administration of stromal cell‐derived factor 1 non‐viral gene therapy in chronic heart failure patients: the STOP‐HF randomized Phase

[89] Gao LR, Chen Y, Zhang NK, Yang XL, et al. Intracoronary infusion of Wharton's jelly‐ derived mesenchymal stem cells in acute myocardial infarction: double‐blind, random‐

ized controlled trial. BMC Medicine. 2015;**13**:162. doi: 10.1186/s12916‐015‐0399‐z

[90] Fiarresga A, Mata MF, Cavaco‐Goncalyes S, et al. Intracoronary delivery of human mes‐ enchymal/stromal stem cells: Insight from coronary microcirculation invasive assess‐ ment in a swine model. PLoS ONE. 2015;**10**:e139870. 2015 Jul 10;13:162. doi: 10.1186/

[91] Helseth R, Opstad T, Solheim S, et al. the effect of intracoronary stem cells injection on markers of leukocyte activation in acute myocardial infarction. Cardiology Research.

[92] Eirin A, Zhu XY, Ferguson CM, et al. Intra‐renal delivery of mesenchymal stem cells attenuate myocardial injury after reversal of hypertension in porcine renovascular dis‐

Catheterization and Cardiovascular Interventions. 2014;**83**:E17‐E25

II trial. European Heart Journal. 2015;**36**:2228‐2238

ease. Stem Cell Research & Therapy. 2015;**6**:7

Cardiology. 2013;**29**:441‐447. ID 582527. http://dx.doi.org/10.1155/2013/582527

trial. PLoS ONE. 2014;**9**:Ide101449

242 Stem Cells in Clinical Practice and Tissue Engineering

Heart Journal. 2014;**35**:1263‐1274

Thoracic Surgery. 2014;**18**:61‐66

Reports. 2015;**3**:447‐452

s12916‐015‐0399‐z

2015;**6**:209‐215


**Chapter 12**

**Provisional chapter**

**Landscape of Manufacturing Process of ATMP Cell**

Immune cell therapies have been studied in numerous clinical trials using Advanced Therapy Medicinal Products (ATMP) against a number of diseases having no or inadequate alternative therapies available, for example, various cancer types, cerebral stroke, cardiac infarction, severe autoimmune disorders, or chronic infections. Despite the enormous number of positive observation in *ex vivo* or animal studies, convincing results in clinical studies remain scanty. The chapter presents a survey and reveals that the manufacturing of immune cells especially for clinical trials is until today primarily performed using archaic, scarcely controlled, and incomparable processes and methods. A deeper characterization of *ex vivo* expanded immune cells is urgently needed not only on the level of a few receptors and ligands on the cell surface but also with respect to the ever-contained subtypes in an expanded immune cell population, the pattern of secreted effector molecules, and their

**Keywords:** immune cells, cell therapy, expansion technologies, T cells, TIL, NK cells,

Immune cells have been the key players as well as glamor of active clinical research of the current decade. T-lymphocytes (T cells), tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T cells), natural killer cells (NK cells), mesenchymal stem/stromal cells (MSC) from bone marrow, umbilical cord blood, umbilical cord tissue layers, placenta, and adipose tissues are the main objects studied in immune cell therapies for various diseases. Publications from numerous preclinical studies and developments on isolation, expansion,

**Landscape of Manufacturing Process of ATMP Cell** 

DOI: 10.5772/intechopen.69335

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

**Therapy Products for Unmet Clinical Needs**

amounts over time and influences from *in vivo* components on them.

**Therapy Products for Unmet Clinical Needs**

Ralf Pörtner, Shreemanta K. Parida,

Ralf Pörtner, Shreemanta K. Parida,

http://dx.doi.org/10.5772/intechopen.69335

MSC, GMP production, ATMP

**Abstract**

**1. Introduction**

Christiane Schaffer and Hans Hoffmeister

Christiane Schaffer and Hans Hoffmeister

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

**Provisional chapter**

### **Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs Therapy Products for Unmet Clinical Needs**

**Landscape of Manufacturing Process of ATMP Cell** 

DOI: 10.5772/intechopen.69335

Ralf Pörtner, Shreemanta K. Parida, Christiane Schaffer and Hans Hoffmeister Christiane Schaffer and Hans Hoffmeister Additional information is available at the end of the chapter

Ralf Pörtner, Shreemanta K. Parida,

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.69335

#### **Abstract**

Immune cell therapies have been studied in numerous clinical trials using Advanced Therapy Medicinal Products (ATMP) against a number of diseases having no or inadequate alternative therapies available, for example, various cancer types, cerebral stroke, cardiac infarction, severe autoimmune disorders, or chronic infections. Despite the enormous number of positive observation in *ex vivo* or animal studies, convincing results in clinical studies remain scanty. The chapter presents a survey and reveals that the manufacturing of immune cells especially for clinical trials is until today primarily performed using archaic, scarcely controlled, and incomparable processes and methods. A deeper characterization of *ex vivo* expanded immune cells is urgently needed not only on the level of a few receptors and ligands on the cell surface but also with respect to the ever-contained subtypes in an expanded immune cell population, the pattern of secreted effector molecules, and their amounts over time and influences from *in vivo* components on them.

**Keywords:** immune cells, cell therapy, expansion technologies, T cells, TIL, NK cells, MSC, GMP production, ATMP

#### **1. Introduction**

Immune cells have been the key players as well as glamor of active clinical research of the current decade. T-lymphocytes (T cells), tumor-infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T cells), natural killer cells (NK cells), mesenchymal stem/stromal cells (MSC) from bone marrow, umbilical cord blood, umbilical cord tissue layers, placenta, and adipose tissues are the main objects studied in immune cell therapies for various diseases. Publications from numerous preclinical studies and developments on isolation, expansion,

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

activation, and phenotyping of the different immune cells are increasing exponentially over the last years. Numerous clinical trials have been conducted or are running, evaluating immune cells as novel Advanced Therapy Medicinal Products (ATMP) therapy against number of diseases having no or inadequate alternative therapies available (see **Table 1**) [2]. There is a strong belief that progress for many severe, life-threatening diseases with bad prognosis that happens in various fulminant cancer types, cerebral stroke, cardiac infarction, severe autoimmune disorders, or chronic infections can either be effectively treated or even cured by immune cell therapy when applied optimally in combinations with other conventional therapies. It is not clear if this belief is justified at this juncture with adequate evidence. It is crucial to critically analyze this question for an insight to address the stumbling blocks to make significant advances in this field.

It is important to reflect into the realities of the limited clinical success in contrary to the promising *in vitro* or *ex vivo* findings. All the inadequate, inconsistent clinical outcomes observed using immune cells *in vivo* following *ex vivo* expansion and all the knowledge gained with immune cells in the numerous clinical trials and individual or small clinical studies using hospital exemptions might have to do with underestimating the key issues with respect to identity, quality, potency, and functionality of the used cells. All the characteristics of the cells as well as influences from production technology, characterization methods, etc. are seldom taken into broader account in robust ways before cells are infused as therapeutics. This will

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

http://dx.doi.org/10.5772/intechopen.69335

247

**2. Requirements and existent challenges in producing immune cells** 

are critical determinants for ATMP cell therapy which are enumerated below.

cells should be performed in a defined procedure consistently.

capable of manufacturing individual cell therapeutics as ATMP.

ible, and GMP conform processes. Amounts of 109

for the clinical trials.

Looking on the methods, technologies, specific equipment, and analytical tools, it is obvious that cell expansion technologies suited for producing specific immune cells for individual immune cell therapy are the weak side of the story. The actual knowledge on the potentials of immune cells is so far only scarcely translated into practical technical solutions, broadly available methods, congruent with the capabilities. That is true for standardized and reproducible expansion of defined immune cell preparations as well as for estimates and methods of measuring specific functionalities of an expanded immune cell population against tumor cells, infections, or inflammations. Moreover, there are also missing generally available methods and techniques for fast and precise measurement of homogeneity of a cell population, of characteristics of sub populations, and single cells [10–13]. All these aspects appear to be widely responsible for the limited progress by these very promising new therapies. Several key issues

• Isolation of specific immune cells from blood or tissues and initial expansion steps are performed with broadly differing methods. The procedures need to be better standardized and harmonized with the validation of the composition of the starting populations of immune cells.

• Ideally, expansion of immune cells should be started with a pure population of specific immune cells. When a mixture of cells (like PBMC) or a piece of tissue are taken, isolation of

• Immune cells for individual cell therapies must be produced in standardized, reproduc-

cell population are assumed as a single optimal therapeutic dose. The manufacturing process with newer available technologies for each cell type is yet to be established coherently

• Standardized and consistent production processes need to be dynamically controlled and documented during the entire culturing process. Only a few production technologies are currently on the market fulfilling all of the abovementioned requirements and thus are

to more than 1010 cells of a pure immune

be highlighted in the following sections.

**for cell therapy**

Isolated immune cells consistently display effectiveness against cancer cells, micro-organisms, inhibition of inflammation parameters, etc., in *ex vivo* test systems and often also in *in vivo* mouse models. The enormous number of positive observations are very encouraging and propel the immune cell research field and drives growing numbers of clinical trials. However, convincing results in clinical studies remain scanty over decades despite the wider engagement and R&D investments [3–6]. Several tumor-infiltrating lymphocytes (TIL) as well as treating therapyresistant hematopoietic and solid tumor cancers with specific activated CAR-T cells show longlasting benefits in otherwise grim cases [7, 8]. In cancer of the hematopoietic system, immune cell therapy has demonstrated its real potency and has become an effective standard therapy [9]. Leukemia and similar forms of cancer of the hematopoietic system can be cured with diseasefree survival or without progression in a high percentage through the transplantation of bone marrow cells from healthy and genetically compatible (allogeneic) donors.


Total number of clinical trials worldwide and in several geographical regions as registered on www.clinicaltrials. gov/, accessed on Feb 15, 2017 [1]. TIL: tumor-infiltrating lymphocytes; CAR-T-lymphocytes: chimeric antigen receptor lymphocytes; MSC: mesenchymal stromal cells; BM-MSC: bone marrow MSC; UC-MSC: umbilical cord MSC; AT-MSC: adipose tissue MSC.

**Table 1.** Current ongoing clinical studies with selected immune cells.

It is important to reflect into the realities of the limited clinical success in contrary to the promising *in vitro* or *ex vivo* findings. All the inadequate, inconsistent clinical outcomes observed using immune cells *in vivo* following *ex vivo* expansion and all the knowledge gained with immune cells in the numerous clinical trials and individual or small clinical studies using hospital exemptions might have to do with underestimating the key issues with respect to identity, quality, potency, and functionality of the used cells. All the characteristics of the cells as well as influences from production technology, characterization methods, etc. are seldom taken into broader account in robust ways before cells are infused as therapeutics. This will be highlighted in the following sections.

activation, and phenotyping of the different immune cells are increasing exponentially over the last years. Numerous clinical trials have been conducted or are running, evaluating immune cells as novel Advanced Therapy Medicinal Products (ATMP) therapy against number of diseases having no or inadequate alternative therapies available (see **Table 1**) [2]. There is a strong belief that progress for many severe, life-threatening diseases with bad prognosis that happens in various fulminant cancer types, cerebral stroke, cardiac infarction, severe autoimmune disorders, or chronic infections can either be effectively treated or even cured by immune cell therapy when applied optimally in combinations with other conventional therapies. It is not clear if this belief is justified at this juncture with adequate evidence. It is crucial to critically analyze this question for an insight to address the stumbling blocks to

Isolated immune cells consistently display effectiveness against cancer cells, micro-organisms, inhibition of inflammation parameters, etc., in *ex vivo* test systems and often also in *in vivo* mouse models. The enormous number of positive observations are very encouraging and propel the immune cell research field and drives growing numbers of clinical trials. However, convincing results in clinical studies remain scanty over decades despite the wider engagement and R&D investments [3–6]. Several tumor-infiltrating lymphocytes (TIL) as well as treating therapyresistant hematopoietic and solid tumor cancers with specific activated CAR-T cells show longlasting benefits in otherwise grim cases [7, 8]. In cancer of the hematopoietic system, immune cell therapy has demonstrated its real potency and has become an effective standard therapy [9]. Leukemia and similar forms of cancer of the hematopoietic system can be cured with diseasefree survival or without progression in a high percentage through the transplantation of bone

**Europe total US total China total**

marrow cells from healthy and genetically compatible (allogeneic) donors.

T-lymphocytes 2343 723 116 401 184 TIL 77 27 4 17 0 CAR-T-lymphocytes 170 123 7 40 78 NK cells 407 140 10 49 62 MSC 339 134 34 29 39 BM-MSC 104 46 12 12 7 UC-MSC 54 24 2 0 16 UCB-MSC 7 3 0 0 2 AT-MSC 20 6 1 1 2

Total number of clinical trials worldwide and in several geographical regions as registered on www.clinicaltrials. gov/, accessed on Feb 15, 2017 [1]. TIL: tumor-infiltrating lymphocytes; CAR-T-lymphocytes: chimeric antigen receptor lymphocytes; MSC: mesenchymal stromal cells; BM-MSC: bone marrow MSC; UC-MSC: umbilical cord MSC; AT-MSC:

**Immune cell type Clinical trials in total Open clinical trials Clinical trials**

**Table 1.** Current ongoing clinical studies with selected immune cells.

make significant advances in this field.

246 Stem Cells in Clinical Practice and Tissue Engineering

adipose tissue MSC.

### **2. Requirements and existent challenges in producing immune cells for cell therapy**

Looking on the methods, technologies, specific equipment, and analytical tools, it is obvious that cell expansion technologies suited for producing specific immune cells for individual immune cell therapy are the weak side of the story. The actual knowledge on the potentials of immune cells is so far only scarcely translated into practical technical solutions, broadly available methods, congruent with the capabilities. That is true for standardized and reproducible expansion of defined immune cell preparations as well as for estimates and methods of measuring specific functionalities of an expanded immune cell population against tumor cells, infections, or inflammations. Moreover, there are also missing generally available methods and techniques for fast and precise measurement of homogeneity of a cell population, of characteristics of sub populations, and single cells [10–13]. All these aspects appear to be widely responsible for the limited progress by these very promising new therapies. Several key issues are critical determinants for ATMP cell therapy which are enumerated below.


• To ensure reproducibility of immune cell production for cell therapies, process conditions have to be controlled, evaluated, documented, and validated. Continuous dynamic control of temperature, pH, and pO<sup>2</sup> in the medium during the immune cell expansion process is indispensable. Glucose and lactate concentration as lead substances for substrates and metabolites should also be under steady control during processing.

**3. Techniques and methods used for isolation and expansion of** 

Blood from patients or donors is a convenient starting material for cell purification and expansion. **Table 2** shows the number of different immune cells contained in a 200 ml sample of peripheral blood. A simple gradient centrifugation of a blood sample is normally taken to obtain "peripheral blood mononuclear cells" (PBMC). *Ex vivo* expansion of the total fraction of T cells, NK, and other cell types within PBMC is possible and is the most commonly used technique to provide starting material for subsequent *ex vivo* expansion of the different

Apheresis is a standard practice to obtain a larger number of CD3+ cells as a starting mate-

components of the whole blood with help of the appropriate device and return the rest of the donation to the donor. Standard leukapheresis and use of anti-CD25 magnetic bead resulted

HLA-mismatched GvHD or organ transplantation rejection [18]. Although the apheresis technology is available in clinical research, no advantages have been comparatively established to that of peripheral blood as a source. Routine production of pure immune cell specimen by apheresis is much more expensive (due to the costly procedure and the infrastructure required to carry out the intervention as well as subsequent large amounts of antibodies

**)**

Neutrophils 400–1300 CD11b+CD16+CD66b+ [14, 15] Lymphocytes 200–600 T cells, B cells (CD3−CD19+) [16] T cells, total 100–400 CD3+CD4+CD8+CD56− [17]

T regulatory cells 5–20 CD3+CD4+CD25+CD127− [18, 19] NK cells 16–80 CD3−CD16+CD56+ [20] NKT cells 0.4–5 CD1d+CD4+CD161+ [21]

Monocytes 40–180 CD14+CD16+CD64+ [23]

**Table 2.** Prevalence of immune cells in blood of healthy donors and surface marker normally used for their identification.

CD3+ T cells [24]. Apheresis has the advantage and choice of extracting one or more

CD4+ CD25+ T regulator (Treg) cells that could be expanded 8.3-fold

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

can be used for clinical trials designed to control

http://dx.doi.org/10.5772/intechopen.69335

**Clones of differentiation commonly determined for identification**

CD127+CCR7+CD62L

CD80+CD86+

CD45RO+CCR7+CD62L+

to a target

249

**Reference**

[17]

[17]

[22]

rial for CAR-T cell production with the goal of obtaining a minimum of 0.6 × 109

**immune cells**

immune cell types.

in a yield of 130 × 106

over three weeks before a dose of 1 × 109

**Immune cell types# Number of cells in 200 ml of** 

**human blood sample (×106**

Memory T cells 40–160 CD3+CD56−CD45RA−

Naive T cells 40–160 CD3+CD25+CD45RA+CD45RO−

Dendritic cells 4–12 CD11c+HLA−DR+CD3−CD19−

of 2 × 109


### **3. Techniques and methods used for isolation and expansion of immune cells**

• To ensure reproducibility of immune cell production for cell therapies, process conditions have to be controlled, evaluated, documented, and validated. Continuous dynamic control

is indispensable. Glucose and lactate concentration as lead substances for substrates and

• All immune cells can be expanded in a common basic medium. However, until now supplementing with serum or thrombocyte lysate is still indispensable. For optimized growth over a prolonged period of time and mass production of the different immune cell specimen, supply with nutrients, gases, and supplements should normally take place in a dynamic, homogenous, and stress-free process to maintain differentiation status, phenotype,

• Irradiated feeder cells are used to achieve an initial expansion with low numbers of isolated effector cells and to increase cell expansion when using insufficient technical equipment (e.g., wells or flasks with uncontrolled processing). Appropriate novel technologies can

• Important criteria of immune cells for therapies are a deeper marker profiling and standard estimates for chemokine and cytokine production efficiency (paracrine factors). Both together are decisive measures for the potency and functional power of immune cell populations/subpopulations for the intended effects. Standardized and comparable values as correlate measures should be mandatory for immune cell therapeutics which need to be

• Subpopulations or even monoclonal immune cells can be produced by guiding an *ex vivo* expansion process through specific activation/inhibition/triggering/priming the cells. Re-

tions (hypoxic/normoxic/hyperoxic), flow dynamics of medium have to be explored, com-

• Immune cells for therapies have to fulfill all the conditions as well as national regulatory requirements and international standards for approval for clinical trial use as investigational medicinal product (IMP) and/or market authorization as ATMP. All the manufacturing steps of an individual immune cell preparation must still be performed in a Clean Room A containment for most processes used for production that consist of more or less open steps. • Current international regulatory standards are in process of evolution as ATMP cannot simply follow the standard of a chemical compound which is a fixed inert molecule, whereas a cell is a living dynamic entity with too many variables and dynamic potentials. It is being considered that regulations on individual cell therapeutics might be registered as distinct category and might be oriented on indications. This needs to mature in course of

• Faster scientific progress, earlier availability, access, and affordable prices of immune cell ATMP can only be achieved when advanced production technologies can be utilized or

time to bring ATMP cell therapy for real clinical use in routine practice.

further developed to realize these desired objectives.

metabolites should also be under steady control during processing.

and function of those cells within physiological ranges.

spective coating/fixed antibodies at bioreactor surface, O<sup>2</sup>

possibly eliminate the use of feeder cells.

pared, and optimized for each condition.

established.

in the medium during the immune cell expansion process

concentration in culture condi-

of temperature, pH, and pO<sup>2</sup>

248 Stem Cells in Clinical Practice and Tissue Engineering

Blood from patients or donors is a convenient starting material for cell purification and expansion. **Table 2** shows the number of different immune cells contained in a 200 ml sample of peripheral blood. A simple gradient centrifugation of a blood sample is normally taken to obtain "peripheral blood mononuclear cells" (PBMC). *Ex vivo* expansion of the total fraction of T cells, NK, and other cell types within PBMC is possible and is the most commonly used technique to provide starting material for subsequent *ex vivo* expansion of the different immune cell types.

Apheresis is a standard practice to obtain a larger number of CD3+ cells as a starting material for CAR-T cell production with the goal of obtaining a minimum of 0.6 × 109 to a target of 2 × 109 CD3+ T cells [24]. Apheresis has the advantage and choice of extracting one or more components of the whole blood with help of the appropriate device and return the rest of the donation to the donor. Standard leukapheresis and use of anti-CD25 magnetic bead resulted in a yield of 130 × 106 CD4+ CD25+ T regulator (Treg) cells that could be expanded 8.3-fold over three weeks before a dose of 1 × 109 can be used for clinical trials designed to control HLA-mismatched GvHD or organ transplantation rejection [18]. Although the apheresis technology is available in clinical research, no advantages have been comparatively established to that of peripheral blood as a source. Routine production of pure immune cell specimen by apheresis is much more expensive (due to the costly procedure and the infrastructure required to carry out the intervention as well as subsequent large amounts of antibodies


**Table 2.** Prevalence of immune cells in blood of healthy donors and surface marker normally used for their identification.

needed for purification of specific cell types from the large number of cells). Another study from Germany reported that ~1010 PBMC from leukapheresis result in about 8% of NK cell yield using Clinimacs (Miltenyi) for further GMP therapeutic expansion [20]. Difference between *ex vivo* expanded cells for cytokine-induced cell therapy for hepatocarcinoma from apheresis and PBMC-derived cells has been reported [25], but this needs further evaluation for a comparative conclusion for various cell types. The initial procedure of cell collection may have an influence on the biological effects of the final cell product.

trigger, and/or prime and expand immune cells *ex vivo*. Single T cell clones being trained already *in vivo* against mutated patients' tumor cells or infected cells are normally present in PBMC only in very low numbers, but they can destroy tumor or infected cells manifold [30]. Concerning T cells, identifying and isolation of specific sub-clones and *ex vivo* expansion of those clones should get more attention in the future research. Besides there are strong hints that treatment with immune cells can be successful only when a systemic response of the total immune system in a patient is achieved along with the local response in the lesions [31]. These are extremely important aspects in the context of production technology enabling to manufacture adequate effective immune cell type for optimal treatment of a patient and a holistic analytical follow up of systemic profiling of the patient in order to gain further

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

http://dx.doi.org/10.5772/intechopen.69335

251

In recent times, the isolation of TIL from tissue and/or microenvironment has proven a much more promising way to get access to T cells being already specifically activated by antigens/ epitopes shed from/presented by cells of solid tumors or metastases they are originating from. Generation of TIL seems no longer a problem. Resected parts of tumor tissues taken by tumor biopsies or from the microenvironment of a tumor is the method of choice, and different suited techniques have been published [32, 33]. It is yet not evident whether devices for standardized processing for cell suspension from the initial tumor tissue by mechanical dispersion and/or additional treatment with enzymes are of advantage [34, 35]. Expansion of TIL even in bigger numbers is possible; up to 1010 can be grown in common media supplemented with serum or thrombocyte lysate and specific cytokines (see **Table 4**). Long time expanded TIL often contain CD3+/CD4+/CD8+TIL. Deeper FACS analyses make likely that sub-clones

are contained which are primed and directed against some single mutated clones [30].

Currently, there has been a major interest in genetically manipulated T cells, which can be transduced with chimeric antigen receptors (CAR). These CAR-T cells express this single chain antigen-binding domain (scFv), which ideally binds to a tumor-associated antigen (TAA). The CAR-T cell/tumor-binding reaction induces an activation signal in the T cells strong enough to destroy tumor cells completely, and contrary to normal T cells, the cytotoxic power of CAR-T cells is not suppressed when CAR-T cells are administered *in vivo*. However, CAR-T cells often elicit a dangerous cytokine storm. This potential adverse effect and the difficult complex and costly construction of efficient CAR-T cells seems to inhibit fast progress

Natural killer (NK) cells are getting more and more attention in the ATMP field, fighting cancer and infections since these innate immune cells can be successfully expanded not only in greater numbers but also in high degree of purity [41–43]. In contrast to T cells, NK cells do not feature immunological incompatibility when administered in haploidentical or even allogenic clinical trials. It is, however, important that NK cells in such settings are totally free from T cells. Advanced production technology makes NK cells attractive to use them in broader clinical perspective. Pure NK cells have shown nearly no unwanted side effects in clinical trials even when administered in high doses. The modern production processes deliver NK cells with enhanced functionalities (high cytotoxicity against many cancer cells in *ex vivo* tests, enhanced paracrine production). Pure NK cells can be manufactured in an

insights to iteratively make the process more efficient.

with this promising cell therapy [41–43].

The whole PBMC fraction consists mainly of T cells (naive and memory CD3+/CD56− T cells, mostly of central T memory type). A minor part of the PBMC contained NK cells identified as CD3−/CD56+ cells. Isolation and *ex vivo* expansion of CD3+/CD56− T cells or CD3−/CD56+ NK cells (and even other specific cell types in patients' PBMC) to bigger numbers does not pose a big problem. It is crucial that T cells are only effective against a cancer cell type or an infectious agent when specifically activated prior to infusion. Hence, T cells, NK cells, and other PBMC-derived cells have to be separated in the first instance and then specifically activated/ primed during the expansion process. Presentation of peptides or epitopes from tumor cells or micro-organisms to T cells *ex vivo*, directly or mediated by dendritic cells, and subsequent *ex vivo* expansion was evaluated over prolonged period of time with no or only limited success in *in vivo* efficiency. *Ex vivo* expanded NK cells showed similar negative results, even when applied to patients in greater numbers [26]. It was recognized that (in blood of patients contained) T regulatory cells do strongly inhibit lytic power of T cells and NK cells. However, this seems not the only reason for disappointing results with only T cell or NK cell fractions.

It is being increasingly recognized that particularly in cancer treatment, breakthrough can be achieved with specific activation and expansion of sub-clones of T cells and/or defined NK cells *ex vivo*. The PBMC-derived cells must be specifically stimulated during expansion which can only be realized by sophisticated integration of steps in the process (either positive or negative selection of unwanted cells through antibodies; enhanced growth of cells of interest through speeding up their mitotic division). In case of T cells as much as possible, effector T cell clones should be present at least in the expanded population with capabilities to persist and execute its desired functions [27]. Expanded NK cells should express high cytotoxicity against patient's tumor cells or infected cells. Specific activation has to be induced during the initiation steps by specific coatings of the surface of the culture flasks/container/bioreactor as well as by addition of different cytokines and growth factors in the medium during the subsequent expansion phase [6, 28, 29]. Cultivation of larger numbers of immune cells under those conditions is often challenging and can scarcely be realized in culture flasks.

A general belief is that a basic prerequisite for a therapeutic application of immune cell types is the provision of adequate number of cells. It is assumed that about 1010 of effective immune cells must be used for treating cancer or infections [1]. A tumor or metastasis of 1 cm3 size yields around 109 cells. In the common cytotoxicity assays using effector versus target cells, five to tenfold more effector cells are often needed to destroy one tumor or infected cell. However, such *ex vivo* results might lead into wrong direction. It is crucial that the cells should have strong binding ability and be armed with specific cytotoxicity against the target cells. Meanwhile, it is apparent that it is important to know how to select, activate, trigger, and/or prime and expand immune cells *ex vivo*. Single T cell clones being trained already *in vivo* against mutated patients' tumor cells or infected cells are normally present in PBMC only in very low numbers, but they can destroy tumor or infected cells manifold [30]. Concerning T cells, identifying and isolation of specific sub-clones and *ex vivo* expansion of those clones should get more attention in the future research. Besides there are strong hints that treatment with immune cells can be successful only when a systemic response of the total immune system in a patient is achieved along with the local response in the lesions [31]. These are extremely important aspects in the context of production technology enabling to manufacture adequate effective immune cell type for optimal treatment of a patient and a holistic analytical follow up of systemic profiling of the patient in order to gain further insights to iteratively make the process more efficient.

needed for purification of specific cell types from the large number of cells). Another study from Germany reported that ~1010 PBMC from leukapheresis result in about 8% of NK cell yield using Clinimacs (Miltenyi) for further GMP therapeutic expansion [20]. Difference between *ex vivo* expanded cells for cytokine-induced cell therapy for hepatocarcinoma from apheresis and PBMC-derived cells has been reported [25], but this needs further evaluation for a comparative conclusion for various cell types. The initial procedure of cell collection may

The whole PBMC fraction consists mainly of T cells (naive and memory CD3+/CD56− T cells, mostly of central T memory type). A minor part of the PBMC contained NK cells identified as CD3−/CD56+ cells. Isolation and *ex vivo* expansion of CD3+/CD56− T cells or CD3−/CD56+ NK cells (and even other specific cell types in patients' PBMC) to bigger numbers does not pose a big problem. It is crucial that T cells are only effective against a cancer cell type or an infectious agent when specifically activated prior to infusion. Hence, T cells, NK cells, and other PBMC-derived cells have to be separated in the first instance and then specifically activated/ primed during the expansion process. Presentation of peptides or epitopes from tumor cells or micro-organisms to T cells *ex vivo*, directly or mediated by dendritic cells, and subsequent *ex vivo* expansion was evaluated over prolonged period of time with no or only limited success in *in vivo* efficiency. *Ex vivo* expanded NK cells showed similar negative results, even when applied to patients in greater numbers [26]. It was recognized that (in blood of patients contained) T regulatory cells do strongly inhibit lytic power of T cells and NK cells. However, this seems not the only reason for disappointing results with only T cell or NK cell fractions. It is being increasingly recognized that particularly in cancer treatment, breakthrough can be achieved with specific activation and expansion of sub-clones of T cells and/or defined NK cells *ex vivo*. The PBMC-derived cells must be specifically stimulated during expansion which can only be realized by sophisticated integration of steps in the process (either positive or negative selection of unwanted cells through antibodies; enhanced growth of cells of interest through speeding up their mitotic division). In case of T cells as much as possible, effector T cell clones should be present at least in the expanded population with capabilities to persist and execute its desired functions [27]. Expanded NK cells should express high cytotoxicity against patient's tumor cells or infected cells. Specific activation has to be induced during the initiation steps by specific coatings of the surface of the culture flasks/container/bioreactor as well as by addition of different cytokines and growth factors in the medium during the subsequent expansion phase [6, 28, 29]. Cultivation of larger numbers of immune cells under those

have an influence on the biological effects of the final cell product.

250 Stem Cells in Clinical Practice and Tissue Engineering

conditions is often challenging and can scarcely be realized in culture flasks.

of 1 cm3

size yields around 109

A general belief is that a basic prerequisite for a therapeutic application of immune cell types is the provision of adequate number of cells. It is assumed that about 1010 of effective immune cells must be used for treating cancer or infections [1]. A tumor or metastasis

sus target cells, five to tenfold more effector cells are often needed to destroy one tumor or infected cell. However, such *ex vivo* results might lead into wrong direction. It is crucial that the cells should have strong binding ability and be armed with specific cytotoxicity against the target cells. Meanwhile, it is apparent that it is important to know how to select, activate,

cells. In the common cytotoxicity assays using effector ver-

In recent times, the isolation of TIL from tissue and/or microenvironment has proven a much more promising way to get access to T cells being already specifically activated by antigens/ epitopes shed from/presented by cells of solid tumors or metastases they are originating from. Generation of TIL seems no longer a problem. Resected parts of tumor tissues taken by tumor biopsies or from the microenvironment of a tumor is the method of choice, and different suited techniques have been published [32, 33]. It is yet not evident whether devices for standardized processing for cell suspension from the initial tumor tissue by mechanical dispersion and/or additional treatment with enzymes are of advantage [34, 35]. Expansion of TIL even in bigger numbers is possible; up to 1010 can be grown in common media supplemented with serum or thrombocyte lysate and specific cytokines (see **Table 4**). Long time expanded TIL often contain CD3+/CD4+/CD8+TIL. Deeper FACS analyses make likely that sub-clones are contained which are primed and directed against some single mutated clones [30].

Currently, there has been a major interest in genetically manipulated T cells, which can be transduced with chimeric antigen receptors (CAR). These CAR-T cells express this single chain antigen-binding domain (scFv), which ideally binds to a tumor-associated antigen (TAA). The CAR-T cell/tumor-binding reaction induces an activation signal in the T cells strong enough to destroy tumor cells completely, and contrary to normal T cells, the cytotoxic power of CAR-T cells is not suppressed when CAR-T cells are administered *in vivo*. However, CAR-T cells often elicit a dangerous cytokine storm. This potential adverse effect and the difficult complex and costly construction of efficient CAR-T cells seems to inhibit fast progress with this promising cell therapy [41–43].

Natural killer (NK) cells are getting more and more attention in the ATMP field, fighting cancer and infections since these innate immune cells can be successfully expanded not only in greater numbers but also in high degree of purity [41–43]. In contrast to T cells, NK cells do not feature immunological incompatibility when administered in haploidentical or even allogenic clinical trials. It is, however, important that NK cells in such settings are totally free from T cells. Advanced production technology makes NK cells attractive to use them in broader clinical perspective. Pure NK cells have shown nearly no unwanted side effects in clinical trials even when administered in high doses. The modern production processes deliver NK cells with enhanced functionalities (high cytotoxicity against many cancer cells in *ex vivo* tests, enhanced paracrine production). Pure NK cells can be manufactured in an easy-to-handle closed system as ATMP in clinical settings near to patients [44, 45]. A particular advantage is that mass amounts of individual NK cells can be produced in a relatively inexpensive way due to low costs for selection, medium, activation, compared with other functionalized immune cells. NK cells can be expanded 2000–50,000-fold in designated perfusion bioreactors, whereas that has by far not been achieved in culture flasks. Adjuvant treatment of stem cell-transplanted patients with pure NK cells becomes a common clinical practice. NK cells isolated from donor blood and expanded effectively avoid infections and GvHD when applied immediately following transplantation.

expanded to big numbers of both cell types in the same bioreactor system under totally closed conditions (unpublished results with ZRP meander type bioreactors, Zellwerk, Germany).

Landscape of Manufacturing Process of ATMP Cell Therapy Products for Unmet Clinical Needs

http://dx.doi.org/10.5772/intechopen.69335

253

The reports about the ever-used methods in the clinical trials do not always contain complete descriptions of the processes and show inconsistencies. However, in general, it can be seen that high-fold expansion as well as big numbers of cells have only been achieved when irradiated feeder cells are used. This might be tolerable during a developmental phase of immune cell therapeutics but should be overcome in case of ATMP manufacturing. In ZRP perfusion bioreactors it was shown that individual immune cell specimen can be expanded to therapeu-

T cells are the most powerful immune cells. Despite the immense research on these cells enriching our deeper knowledge into T cell biology as well as their kinetics in health and diseases, the different T cell subsets are yet not routinely used as cell therapeutics. This is at least partly due to the complex nature of immune cells and many unsolved technical problems to produce and handle them. In **Tables 3**–**5**, some parameters have been compiled giving insight into methods and technologies as well as expansion success and purity of the different T cell types being used in clinical trials. The chosen examples in the tables are representative of the field and enlighten the diversity of production processes and produced cell specimen, and it explains probably in part the inconsistent and predominantly unsatisfying clinical results

Vaccination with dendritic cells being *ex vivo* treated with proteins or peptides from tumor cells or micro-organisms as well as present target-specific peptides to T cells *ex vivo*, directly

> **Expansion by x-fold**

Flask 42 n.a. n.a. n.a. [59]

cells

Wave system 13 101 1.37 × 1011 T

PBMC Flask 14 169 33 × 108 62% [58]

**Cell harvest Cell purity (%) Reference**

98.5% CD3+ [27]

**4. Quantity and quality of T cells, TIL, and CAR-T used in** 

**Cultivation time (days)**

**Table 3.** Manufacturing processes of T cells in clinical trials (n.a.= not available).

tic amounts without irradiated feeder cells.

with more or less identical cell specimens.

**Expansion device used**

**Cell source; supplements; activation**

PBMC/apheresis isolated by αCD3+ coated dynabeads, exp with IL2/αCD28

PBMCs/apheresis CD25+Tregs depl, Stim with autol DC pulsed MART peptides, suppl IL2, IL7, IL21, CD8+ CTL sorted, exp Rapid Exp Prot

**acknowledged clinical trials**

Common sources for isolation of MSC are bone marrow aspirate, cord blood, and pieces of umbilical cord/placenta tissue/adipose tissue. MSC were originally identified in the 1970s from cellular suspensions from spleen and bone marrow by their capacity to adhere to plastic—which is still the standard form to culture MSCs and also by their ability to form colonies from single cells (explanted *ex vivo*), their fibroblast-like appearance and their capacity to differentiate into fat, cartilage, and bone. MSC are defined by surface markers of CD105, CD90, and CD73 expression, yet not CD45, CD34, and CD14 as of the consensus of ICSCT working group [46]. Recently, ICSCT also have defined a broad consensus of the international standards for harmonized potency assays to boost the clinical development of ATMP MSC therapy for many unmet clinical needs despite different tissue sources and disparate culture expansion protocols. Three preferred analytic methods in a matrix assay approach, namely, quantitative RNA analysis of selected gene products; flow cytometry analysis of functionally relevant surface markers, and protein-based assay of secretome have been proposed to reflect on the immunomodulatory potential of the ATMP cells for different clinical therapeutics as well as to evolve the regulatory landscape for the sake of the progress in the field [47].

Several techniques are employed for liposuction used for adipose tissue-derived stromal cell collections [48]. Processed lipoaspirate (LPA) contains multipotent cells that can be an alternate stem cell source to bone-marrow-derived MSCs. LPA contains stromal vascular fraction (SVF) containing a number of different cell types such as adipose stromal cells (ASC), pericytes, endothelial cells, fibroblasts, preadipocytes, and hematopoietic stem cells. ASC have differentiation potential to myogenic, osteogenic, chondrogenic, or adipogenic on culturing with specific induction media [49]. SVF contains a lot of vascular cells and hematopoetic cells that have to be eliminated before expansion of remaining MSC.

For the isolation of MSC adherence of these cells, plastic surfaces are used. The usual procedure is to put the starting material into culture flasks or discs. After 10–20 days, nonadherent cells are washed out, and the adhered MSC colonies are passaged, suspended in a fresh medium, and seeded in new flasks for further expansion. Thus, expanded MSC have been used in all clinical trials (see **Table 7**). The unavoidable detaching procedures of MSC at passaging influence the receptor quality of MSC. Long-term cultivation of big numbers of MSC in bioreactors is possible and the provision of large seeding areas avoids unwanted differentiation of expanded MSC [54–57]. Procedures have been worked out to proceed directly into the expansion phase of MSC. Outgrowth and isolation of MSC can be successfully performed by giving BM aspirate into the sterile plastic vessels of perfusion bioreactors. MSC from BM aspirate are not only diverse tumor tissue preparations but can also be placed directly in ZRP meander perfusion bioreactors. MSC colonies or outgrown TIL from tumor tissue pieces can then further be expanded to big numbers of both cell types in the same bioreactor system under totally closed conditions (unpublished results with ZRP meander type bioreactors, Zellwerk, Germany).

The reports about the ever-used methods in the clinical trials do not always contain complete descriptions of the processes and show inconsistencies. However, in general, it can be seen that high-fold expansion as well as big numbers of cells have only been achieved when irradiated feeder cells are used. This might be tolerable during a developmental phase of immune cell therapeutics but should be overcome in case of ATMP manufacturing. In ZRP perfusion bioreactors it was shown that individual immune cell specimen can be expanded to therapeutic amounts without irradiated feeder cells.
