Preface

Chapter 7 **Umbilical Cord Blood-Derived Therapies as a Treatment for**

Chapter 8 **Optimization of Unrelated Donor Cord Blood Transplantation for Thalassemia: Implications for Other Non‐Malignant Indications such as HIV Infection or Autoimmune**

**Section 4 Tissue Engineering - Biomaterials and Cell-Based Therapies Derived from Umbilical Cord Blood 219**

Chapter 9 **Myoblast Differentiation of Umbilical Cord Blood Derived Stem Cells on Biocompatible Composites Scaffold Meshes 221**

Richard Duggleby, Steve Cox, J Alejandro Madrigal and Aurore

Christine Chow, Tracie Dang, Vincent Guo, Michelle Chow, Qingyu Li, Delon Te‐Lun Chow, Elizabeth Rao, Tony Zeng, Baixiang Wang

**Graft-Versus-Host Disease 169**

Saudemont

**VI** Contents

**Diseases 181**

and Robert Chow

Biswadeep Chaudhuri

The present book includes four sections entitled (1) Umbilical Cord Blood Banking and Processing, (2) Umbilical Cord Blood: Clinical Analysis, (3) Clinical Applications of the Um‐ bilical Cord Blood, and (4) Tissue Engineering: Biomaterials and Cell-Based Therapies De‐ rived from the Umbilical Cord Blood.

Umbilical cord blood (UCB) and, more recently, umbilical cord tissue (UCT) have been stor‐ ed cryopreserved in private and public cord blood and tissue banks worldwide in order to obtain hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs), and although guidelines exist that imply high-quality standards and total rastreability of the units (Net‐ Cord—Foundation for the Accreditation of Cellular Therapy), standardized procedures for UCB and UCT transport from the hospital/clinic to the laboratory, storage, processing, cryo‐ preservation, and thawing are still awaited. These may be critical in order to obtain higher viable stem cell number after thawing and to limit microbiological contamination. The isola‐ tion and culture of MSCs from UCB and Wharton's jelly or UCT have been performed by us and other research groups all over the world in order to obtain MSCs to be used in clinical applications in regenerative medicine. Also the laboratory processing and cryopreservation protocols of the UCB and UCT units following the recommended technical procedures by NetCord and national health authorities like the ones adopted by Biosckin, Molecular and Cell Therapies, SA, in Portugal, have been validated, and the recovery and viability rate of the cryopreserved stem cells after thawing are already high, clearly demonstrating the posi‐ tive knowledge and technical improvement in this area of research.

The MSCs from the UCT and UCB and the HSCs from the UCB have been used in several clinical trials in children and adults, concerning a wide range of pathologies and diseases, for instance, for the treatment of cerebral paralysis, that can be daily consulted, for instance, in www.clinicaltrials.com. The MSCs have also been intensively studied to promote engraft‐ ment in allogenic hematopoietic stem cell transplantation, as a matter of fact, the MSCs, in particularly the ones isolated from the Wharton's jelly, are nowadays used as a coadjuvant in hematopoietic treatments using UCB and bone marrow transplantation. Nowadays, the cryopreservation of UCB and UCT is performed worldwide in private and public cord blood banks, since the umbilical cord blood was used for the first time in a child with Fanconi anemia with his HLA-identical sibling, in hematopoietic treatments for blood disorders and hemato-oncological diseases. Also the cotransplantation of MSCs and hematopoietic stem cells has a positive clinical outcome in hematological malignancy patients.

UCB is an alternative source of HSCs, when compared with bone marrow and peripheral blood after apheresis, especially for patients requiring allogenic HSC transplantation but lacking a suitable human leukocyte antigen (HLA)-matched donor. Using allogenic cord

blood (CB) has many advantages, including lower HLA-matching requirements and in‐ creased donor availability. Also, HSCs from the UCB have higher proliferative capacity and decreased immune reactivity, so lower rates of graft-versus-host disease are observed. On the other hand, using autologous CB, the HLA matching is complete, and the CB unit is im‐ mediately available for clinical use. Furthermore, with over 650,000 cryopreserved CB units currently stored in international CB banks worldwide, CB is rapidly available for those pa‐ tients requiring transplantation, avoiding the time-consuming search for a histocompatible donor of bone marrow or CB unit from a public bank. However, concern remains over the fact that in some CB units, there is a low HSC dose available, resulting in delayed engraft‐ ment and poor immune reconstitution. For instance, the best UCB units should contain more than 2 x 107 nucleated cells/kg and more than 2x105 CD34+ cells/kg, and the number of HLA mismatches should not be superior to 2 if the option is a CB allogenic unit. Further research is currently undertaken to improve the results of allogenic and autologous hematopoietic stem cell transplants and includes the use of double or sequential cord blood transplants, the improvement of the preparative regimen with nonmyeloablative drugs, the ex vivo ex‐ pansion of progenitor cells, and the use of MSCs as coadjuvants for the HSCs transplants. The therapeutic dosage of MSCs commonly employed for infusion in treatment of graft-ver‐ sus-host disease is more than 2 x 106 /kg body weight of the patient, the ex vivo expansion of MSCs for therapeutic applications is necessary, and this concern also includes the cell-based therapies in regenerative medicine.

Human MSCs can influence tissue regeneration and scar tissue formation processes mainly by their paracrine effect through a range of biomolecules synthesized by these cells, more than their direct differentiation into functional tissue. The niche created by the expression of chemotactic factors attracts endogenous MSCs to the area of injury. Although transplanted MSCs do not persist alive in the graft environment for a long period, they are able to initiate the formation of this niche in the injured environment, promoting the mobilization of en‐ dogenous stem/progenitor cells to the site of injury. Some authors observed that the engraft‐ ed cells disappeared rapidly (being susceptible to death by pro-inflammatory cytokines and reactive oxygen species) and at that moment there was a correspondent increase in the num‐ ber of host cells to the area of injury. They also described the modulation and recruitment of host cells both locally and systemically in response to exogenous MSC engraftment. These transplanted MSCs also have the value of acting as "protectors" to other cell types. Under‐ standing the role of the various mechanisms involved in the environment of these stem cell "niches" is extremely important not only to understand the concept of stem cell biology but also for the establishment of in vitro culture protocols meant for biomedical use. It is becom‐ ing particularly relevant to the detailed characterization of MSCs secretome, as the factors secreted by these cells may be the main effectors of their therapeutic action. The low surviv‐ al rate of transplanted cells into the damaged tissue has been proved in several pathologies and, consequently, the clinical benefits are only transient and attributed mostly to trans‐ planted cell-associated paracrine effects that, for example, in injured myocardium, stimulate angiogenesis by stimulating endothelial cell adhesion through chemotactic factors. This re‐ cent paradigm has suggested that the biomolecules synthesized by stem cells may be as im‐ portant, if not more so, than differentiation of the cells in eliciting functional tissue repair. This evidence suggests that the culture medium obtained from in vitro culture and expan‐ sion of MSCs and HSCs (CD34+ cells) or the umbilical cord blood serum/plasma are proba‐ bly better therapeutic options compared to the in vivo transplantation of these stem cells, as it can benefit from the local tissue response to the secreted molecules without the difficulties and complications associated to the engraftment of the allotransplanted cells. In addition to immunoregulatory, pro-angiogenic, and antiapoptotic factors, MSCs also secrete neurotro‐ phic factors, which could potentially be used in neurological disorders.

blood (CB) has many advantages, including lower HLA-matching requirements and in‐ creased donor availability. Also, HSCs from the UCB have higher proliferative capacity and decreased immune reactivity, so lower rates of graft-versus-host disease are observed. On the other hand, using autologous CB, the HLA matching is complete, and the CB unit is im‐ mediately available for clinical use. Furthermore, with over 650,000 cryopreserved CB units currently stored in international CB banks worldwide, CB is rapidly available for those pa‐ tients requiring transplantation, avoiding the time-consuming search for a histocompatible donor of bone marrow or CB unit from a public bank. However, concern remains over the fact that in some CB units, there is a low HSC dose available, resulting in delayed engraft‐ ment and poor immune reconstitution. For instance, the best UCB units should contain more

mismatches should not be superior to 2 if the option is a CB allogenic unit. Further research is currently undertaken to improve the results of allogenic and autologous hematopoietic stem cell transplants and includes the use of double or sequential cord blood transplants, the improvement of the preparative regimen with nonmyeloablative drugs, the ex vivo ex‐ pansion of progenitor cells, and the use of MSCs as coadjuvants for the HSCs transplants. The therapeutic dosage of MSCs commonly employed for infusion in treatment of graft-ver‐

MSCs for therapeutic applications is necessary, and this concern also includes the cell-based

Human MSCs can influence tissue regeneration and scar tissue formation processes mainly by their paracrine effect through a range of biomolecules synthesized by these cells, more than their direct differentiation into functional tissue. The niche created by the expression of chemotactic factors attracts endogenous MSCs to the area of injury. Although transplanted MSCs do not persist alive in the graft environment for a long period, they are able to initiate the formation of this niche in the injured environment, promoting the mobilization of en‐ dogenous stem/progenitor cells to the site of injury. Some authors observed that the engraft‐ ed cells disappeared rapidly (being susceptible to death by pro-inflammatory cytokines and reactive oxygen species) and at that moment there was a correspondent increase in the num‐ ber of host cells to the area of injury. They also described the modulation and recruitment of host cells both locally and systemically in response to exogenous MSC engraftment. These transplanted MSCs also have the value of acting as "protectors" to other cell types. Under‐ standing the role of the various mechanisms involved in the environment of these stem cell "niches" is extremely important not only to understand the concept of stem cell biology but also for the establishment of in vitro culture protocols meant for biomedical use. It is becom‐ ing particularly relevant to the detailed characterization of MSCs secretome, as the factors secreted by these cells may be the main effectors of their therapeutic action. The low surviv‐ al rate of transplanted cells into the damaged tissue has been proved in several pathologies and, consequently, the clinical benefits are only transient and attributed mostly to trans‐ planted cell-associated paracrine effects that, for example, in injured myocardium, stimulate angiogenesis by stimulating endothelial cell adhesion through chemotactic factors. This re‐ cent paradigm has suggested that the biomolecules synthesized by stem cells may be as im‐ portant, if not more so, than differentiation of the cells in eliciting functional tissue repair. This evidence suggests that the culture medium obtained from in vitro culture and expan‐ sion of MSCs and HSCs (CD34+ cells) or the umbilical cord blood serum/plasma are proba‐ bly better therapeutic options compared to the in vivo transplantation of these stem cells, as it can benefit from the local tissue response to the secreted molecules without the difficulties

CD34+ cells/kg, and the number of HLA

/kg body weight of the patient, the ex vivo expansion of

than 2 x 107 nucleated cells/kg and more than 2x105

sus-host disease is more than 2 x 106

VIII Preface

therapies in regenerative medicine.

Considering the worldwide availability of UCB and UCT units and the absence of ethical concerns, UCB and UCT will probably become important and best sources for cell-based therapies for hematological and nonhematological pathologies. The UCB will also have a crucial role in neonatology-predictive analysis in the near future.

#### **Ana Colette Maurício, DVM, PhD**

Associate Professor in Habilitation Abel Salazar Biomedical Sciences Institute from the University of Porto (ICBAS-UP) Porto, Portugal

**Umbilical Cord Blood Banking and Processing**

#### **Cord Blood Stem Cell Processing, Banking and Thawing Cord Blood Stem Cell Processing, Banking and Thawing**

Robert Y.K. Chow, Qingyu Li, Christine Chow, Vincent Guo, Tracie Dang, Andrew Rao, Tony Zeng, Delon Te-Lun Chow, Baixiang Wang and Michelle Chow Robert Y.K. Chow, Qingyu Li, Christine Chow, Vincent Guo, Tracie Dang, Andrew Rao, Tony Zeng, Delon Te-Lun Chow, Baixiang Wang and Michelle Chow

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

http://dx.doi.org/10.5772/65033

#### **Abstract**

Unrelated donor cord blood (CB) is one of the three sources of hematopoietic stem cell transplantation (HSCT) that are capable of curing ~80–160 standard hematologic and certain non-hematologic indications. Despite its many advantages, the principal drawback for CB in HSCT is its limited cell dose. Our group has focused on developing minimally manipulated technologies and strategies to maximize stem, progenitor, and nucleated cell doses to overcome this limitation. The term "MaxCell" is used in this chapter to denote two proprietary CB volume reduction processing technologies that yield virtually 100% recovery of all cell lineages in the manufactured CB products, including what the authors designate as "second generation" (2nd Gen) or plasma depletion/reduction (PDR) and "third generation" (3rd Gen) MaxCB or MaxCord CB processing technologies. In our proposed nomenclature system, the traditional red cell reduction (RCR) processing techniques are designated as "first generation" methods. The properties of various popular 1st Gen techniques are compared to the MaxCell CB processing technologies. Parallel processing with the traditional hetastarch (HES) RCR technique and the patented MaxCell CB processing technology were used to compare recovery of the various stem, progenitor, nucleated, and red cell lineages. MaxCell processing technology achieved virtually 100% recovery of all stem, progenitor, and nucleated cells tested after processing, with high cell viability upon thawing. The higher cell recovery produced MaxCell inventory with higher average stem, progenitor and nucleated cell doses, allowing patients to receive CB products with higher cell doses. Clinical outcome of HSCT using MaxCell CB products was compared to the outcome of HSCT with RCR CB products published in the literature from transplant data registries or CB banks. To allow for more rigorous comparisons, two matched-pair analysis (MP) were performed using a logistic regression model to find pairs of pediatric patients with hematologic malignancies and thalassemia transplanted with RCR CB or

© 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. © 2017 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.

MaxCell CB, and patients receiving MaxCell CB showed superior engraftment, survival, and transplant-related mortality, confirming pre-match observations.

In addition, the three main post-thaw manipulation methods are reviewed. Comparison data for some of the thaw methods are presented, and matched-pair analysis was used to confirm the superiority of direct infusion thaw method over post-thaw wash for MaxCell CB products.

**Keywords:** cord blood banking, cord blood processing, MaxCell cord blood processing, MaxCB cord blood processing, stem cell processing, cord blood thawing, cord blood cryopreservation, cord blood transplantation, 2nd and 3rd generation cord blood technologies

#### **1. Introduction**

Today, hematopoietic stem cell transplantation (HSCT) can be performed using stem cells derived from three sources: bone marrow (BM), peripheral blood (PB), and umbilical cord blood (CB) from autologous, related allogeneic, or unrelated allogeneic donors. Autologous HSCT is not indicated for most indications, and only about one-third of patients in need of HSCT have a suitable related donor. Although there are currently tens of millions of adult volunteer donors registered worldwide, about 60% of patients will not find a suitably HLAmatched unrelated adult donor, and thus cannot access HSCT [1]. For minority patients, the probability of finding an HLA-matched unrelated adult donor is further reduced. Since the search process for an adult donor often takes several months, a significant proportion of patients will become higher risk or ineligible for HSCT or even die while waiting for the donor search [2]. In contrast, without the possibility of last minute donor refusal, CB products are available upon request and can be shipped to any transplant center in the world immediately. Importantly, cord blood transplantation (CBT) is associated with a lower incidence and severity of graft-versus-host disease (GvHD), and a partial HLA match between the donor and recipient is tolerable [3], making it an ideal donor source for minority patients without large adult donor registries.

Compared with historical controls of BM or PB HSCT, CBT has shown favorable clinical outcome despite significantly worse HLA match [3]. In the pediatric setting, CBT can be now considered established practice. For adults, CBT adoption has been slower because of cell dose limitation. Many strategies focused on overcoming this limitation are being developed, including double CBT, the utilization of two CB unit grafts [4–6], the combination of an unrelated CB graft with haplo-identical donor stem cells [7], and foregoing the post-thaw wash with direct infusion or reconstitution/dilution, which have shown promising results [8, 9]. Engraftment and survival appear to be at least equivalent to or may even be superior to historical data using post-thaw wash for CB products versus when CB was not washed. One area which our group has focused on has been the optimization of the entire CB banking process, and in particular, the CB processing and thaw manipulations.

Almost all the published literature on CB processing has shown significant cell losses with various volume-reduction red cell reduction (RCR) processing techniques (referred to in this chapter as first generation or 1st Gen), such as the hydroxyethyl starch (hetastarch or HES) sedimentation technique, Optipress II, AXP-Express, PrepaCyte, Sepax etc.,…, with the range of loss generally around 25% for total nucleated cells (TNC) and with commensurate or higher CD34+ progenitor and colony-forming unit (CFU) loss [10–17]. As this is one of the areas that CB banks (CBBs) can have an impact, Chow has developed two novel MaxCell CB processing technologies to improve cell doses of CB products—plasma depletion/reduction processing without the reduction of red blood cells (RBC) or second generation (2nd Gen) [18] and the MaxCord or MaxCB third generation (3rd Gen) technology which combines the advantages of 1st Gen and 2nd Gen, without any of the associated disadvantages. While the processing methods and cryopreserved products are distinctly different, the cellular composition of the final infused product is identical between the two MaxCell technologies in the preferred embodiment of 3rd Gen.

Another area which has significant impact on CB potency and clinical outcome is the thaw and post-thaw manipulations of CB products. Transplant centers which do not follow CBB's prescribed and validated procedure for thawing risk decreased viability of the CB product for their patients, delayed engraftment, graft failure, transplant-related mortality, and the potential for severe adverse events (SAE) in rare cases due to thaw and Dimethyl sulfoxide (DMSO) toxicity mediated release of free hemoglobin, red cell and white cell lysates, chemokines and cytokines that may cause hemodynamic destabilization. Some of the recent development and data are reviewed.

While every step from collection to infusion of CB impacts engraftment and ultimately patient survival, in this chapter, we will focus on the roles of processing and post-thaw manipulations.
