**5. Potential application of placenta-derived stem cells to CF**

#### **5.1. Placenta-derived stem cells for lung diseases**

The first report demonstrating a therapeutic effect of placenta-derived stem cells in lung diseases is that by Cargnoni and colleagues [172]. In a mouse model of bleomycin-induced lung injury, transplantation of fetal membrane-derived cells resulted in a reduction in the severity of pulmonary fibrosis. This result was obtained when cells were administered either systemically (intravenous or intraperitoneal) or locally (intratracheal) 15 min after intratra‐ cheal bleomycin instillation and in two different settings, *i.e.* either using allogeneic or xenogeneic (a mixture of 50% human amnion/chorion mensenchymal stem cells and 50% hAEC) cells. Although the inflammatory score was not decreased, a reduction in the number of infiltrating neutrophils was observed. It is worth noting that that the presence of neutrophils is known to be associated with poor prognosis in idiopathic pulmonary fibrosis in humans [173]. The question arises whether these anti-inflammatory and anti-fibrotic effects may be due to the engraftment of placenta-derived stem cells or to the secretion of soluble factors. In this study allogeneic or xenogeneic cells were detected in the injured lung of transplanted mice, although not in a quantitatively fashion, by means of PCR analysis, and these results are in accordance with those obtained by Bailo and colleagues, who demonstrated microchimerism upon transplantation of human amnion and chorionic cells in neonatal swine and rats [152]. The release of soluble factors has been addressed in a further study. The administration of conditioned medium generated from hAMSC to bleomycin-treated mice determined a reduction in lung fibrosis scores in terms of fibrosis distribution, fibroblast proliferation, collagen deposition and alveolar obliteration [174]. This study support the increasing evidence that MSC isolated from various sources produce bioactive molecules, so that injection of conditioned medium obtained from MSC could be an effective experimental treatment for different tissue injuries [175, 176]. Further studies are therefore warranted to elucidate the mechanisms of action of placenta-derived cells in this model, in particular paracrine factors that act to down-regulate neutrophil recruitment.

It has to be said that the role of exogenous stem cells in pulmonary fibrosis is controversial, meaning that some studies have demonstrated that these cells can act as a potential source of fibroblast, which may accentuate the fibrotic process [177]. Since these findings were obtained with BM-derived stem cells, it should be further assessed if a similar behaviour is presented by amniotic-derived stem cells. Of note, placenta-derived cells did not exert any profibrotic effect after their transplantation [172].

*In vitro* studies have so far demonstrated that co-cultures of hAMSC and CF epithelial cells originated from bronchi can elicit CFTR protein expression in 33-50% hAMSC, in front of 6% prior to the co-cultures, and the lower the hAMSC:CFBE41o- ratios the lower the CFTR expression in hAMSC [136]. Indirect co-cultures data indicate that this effect is primarily due to the contact between hAMSC and epithelial cells, and not due to factors acting by a paracrine manner. BM-MSC acquired an airway epithelium phenotype when co-cultured with respira‐ tory epithelial cells and determined a partial resumption of the chloride secretion defect in CF epithelia [178]. Preliminary analysis of the chloride transport defect in co-cultures between CF cells and hAMSC showed a partial correction of the chloride efflux (Carbone et al., unpublished results). Furthermore, since only 6-20% of corrected cells is needed to revert the basic defect in chloride secretion [179], our data showing that 33-50% of hAMSC acquired CFTR expression shed a positive light on the use of amnion MSCs in the CF treatment. Overall, these data point out to a cross talk between amniotic and epithelial cells, for which a critical number of hAMSC is needed. Indeed, in other co-culture systems, developed with MSC and chondrocytes, it has been shown universally that the more chondrocytes the lower the expression of extracellular matrix genes and functional properties of engineered cartilage [180, 181]. Since the cellular interactions between epithelial and mesenchymal cells in monolayer co-culture are likely to be bi-directional, a possible mode of action could be cross talk between cells via gap junctions, which has been observed *in vivo* in the lung between transplanted MSC and resident epithelial cells [182].

Overall, the potential usefulness of placenta-derived stem cells in CF lung disease might be either in the correction of the early basic defect (chloride transport) or in late remodelling events (pulmonary fibrosis).

#### **5.2. Placenta-derived stem cells for liver diseases**

Several preclinical studies have reported to date that placenta-derived stem cells can engraft into the liver and perform hepatic functions *in vivo*. Takashima and colleagues [183] showed that after transplantation of human amniotic membrane into the peritoneum of SCID mice, human albumin could be detected in the sera and peritoneal fluid of these animals from day 1 until day 7. Sakuragawa and colleagues [184] showed that the transplantation of hAEC transduced with the β-galactosidase gene into the livers of SCID mice resulted in detection of β-galactosidase-positive cells at 1 week after transplantation, indicating that the transplanted cells had been integrated into the hepatic parenchyma within a few days [184]. More recently, it has been shown that six months after transplantation of hAEC into the livers of SCID/beige mice that had been pretreated with retrorsine, most mature liver genes were expressed at levels comparable to those of authentic human adult livers, including the major CYP genes, other metabolic enzymes, plasma proteins, and hepatocyte-enriched transcription factors and genes encoding hepatic-transporter proteins [185].

These studies provide compelling evidence in support of the functional hepatic potential of hAEC *in vivo*, thereby supporting the potential of hAEC as a useful tool for liver regeneration in the future.

with BM-derived stem cells, it should be further assessed if a similar behaviour is presented by amniotic-derived stem cells. Of note, placenta-derived cells did not exert any profibrotic

*In vitro* studies have so far demonstrated that co-cultures of hAMSC and CF epithelial cells originated from bronchi can elicit CFTR protein expression in 33-50% hAMSC, in front of 6% prior to the co-cultures, and the lower the hAMSC:CFBE41o- ratios the lower the CFTR expression in hAMSC [136]. Indirect co-cultures data indicate that this effect is primarily due to the contact between hAMSC and epithelial cells, and not due to factors acting by a paracrine manner. BM-MSC acquired an airway epithelium phenotype when co-cultured with respira‐ tory epithelial cells and determined a partial resumption of the chloride secretion defect in CF epithelia [178]. Preliminary analysis of the chloride transport defect in co-cultures between CF cells and hAMSC showed a partial correction of the chloride efflux (Carbone et al., unpublished results). Furthermore, since only 6-20% of corrected cells is needed to revert the basic defect in chloride secretion [179], our data showing that 33-50% of hAMSC acquired CFTR expression shed a positive light on the use of amnion MSCs in the CF treatment. Overall, these data point out to a cross talk between amniotic and epithelial cells, for which a critical number of hAMSC is needed. Indeed, in other co-culture systems, developed with MSC and chondrocytes, it has been shown universally that the more chondrocytes the lower the expression of extracellular matrix genes and functional properties of engineered cartilage [180, 181]. Since the cellular interactions between epithelial and mesenchymal cells in monolayer co-culture are likely to be bi-directional, a possible mode of action could be cross talk between cells via gap junctions, which has been observed *in vivo* in the lung between transplanted MSC and resident epithelial

Overall, the potential usefulness of placenta-derived stem cells in CF lung disease might be either in the correction of the early basic defect (chloride transport) or in late remodelling

Several preclinical studies have reported to date that placenta-derived stem cells can engraft into the liver and perform hepatic functions *in vivo*. Takashima and colleagues [183] showed that after transplantation of human amniotic membrane into the peritoneum of SCID mice, human albumin could be detected in the sera and peritoneal fluid of these animals from day 1 until day 7. Sakuragawa and colleagues [184] showed that the transplantation of hAEC transduced with the β-galactosidase gene into the livers of SCID mice resulted in detection of β-galactosidase-positive cells at 1 week after transplantation, indicating that the transplanted cells had been integrated into the hepatic parenchyma within a few days [184]. More recently, it has been shown that six months after transplantation of hAEC into the livers of SCID/beige mice that had been pretreated with retrorsine, most mature liver genes were expressed at levels comparable to those of authentic human adult livers, including the major CYP genes, other metabolic enzymes, plasma proteins, and hepatocyte-enriched transcription factors and genes

effect after their transplantation [172].

16 Regenerative Medicine and Tissue Engineering

cells [182].

events (pulmonary fibrosis).

**5.2. Placenta-derived stem cells for liver diseases**

encoding hepatic-transporter proteins [185].

MSC represent an alternative tool for the establishment of a successful stem-cell-based therapy of liver diseases [186] with preliminary clinical improvements in acute and chronic hepatic diseases [187, 188]. To date, several studies on animal models reported the beneficial effects of MSC in promoting hepatic tissue regeneration [189]. Overall, a number of different mecha‐ nisms contribute to the therapeutic effects exerted by MSC, among which their differentiation into functional hepatic cells. However, these studies have not provided definitive evidence that MSC have a capability to differentiate into functional hepatocytes *in vivo* [190]. Rather, the observed improvements could be attributed to the known property of MSC to produce a series of growth factors and cytokines that could suppress inflammatory response, reduce hepato‐ cytes apoptosis, regress liver fibrosis, and enhance hepatocytes functionality [191, 192].

Although numerous studies have reported that BM-derived MSC can reduce carbon tetrachloride (CCl4)-induced liver fibrosis in mice, the mechanism by which MSC repair the fibrosis is unclear, and the results are controversial [190, 193-197]. One possibility is that MSC differentiate into hepatocytes, because of the *in vivo* niche, and secrete growth factors that promote liver regeneration. Another possibility is that MSC suppress hepatic stellate cells activity and secrete metalloproteinases (MMPs), thereby eliminating deposi‐ tion of extracellular matrix [198]. It has been demonstrated that fibrosis, infiltration of neutrophils, synthesis of collagen I and α-smooth muscle actin (α-SMA), and expression of inflammatory were all reduced by infusion of isogenic MSC [199]. It is possible that these responses were partly due to the upregulation of cytoglobin expression by hepatic stellate cells, which protect against oxidative stress and controls tissue fibrosis and at the same time inhibits the activation of those cells to become myofibroblasts [200]. Finally, it has been demonstrated that intravenous administration of MSC caused an increase in IL-10 mRNA in the liver and protein in the blood in a CCl4-induced liver fibrosis rat model [201]. IL-10 is an inhibitor of many cytokines that stimulate liver fibrosis, such as IL-6, TNF-α and TGF-β, all downregulated by the MSC infusion. In addition, IL-10 can suppress tissue inhibitor of metalloproteinase (TIMP)-1 expression and thereby relieve MMP-1 to de‐ grade liver collagen deposits [202, 203].

In a recent study, hAMSC were infused in mice with CCl4-induced hepatic cirrhosis and exerted various beneficial effects such as reduction of hepatic stellate cell activation, decrease of hepatocyte apoptosis, and reduction of hepatic fibrosis [204]. Infusion of hAMSC also depressed hepatocyte senescence and resulted in engraftment of hAMSC into the host liver as judged by the expression of the hepatocyte-specific markers, human albumin and α-fetopro‐ tein. Finally, a study demonstrated that human AM, when applied as a patch onto the liver surface, reduced progression of experimental biliary fibrosis induced in rats by the biliary duct ligation procedure [205]. Again, a beneficial effect related to the release of soluble factors by the human AM patch has been invoked, since no massive (or at least very low/undetectable) engraftment of AM-derived cells occurred in the host liver.

#### **6. Conclusion**

Placenta-derived stem cells are endowed with interesting features that are important for choosing them as a source for approaches aimed to regenerative medicine: immune-privileged status, secretion of biomolecules with anti-scarring and anti-inflammatory properties, and, least but not last, no ethical concerns. Although the AM and AM-derived stem cells have been used in the clinics for over one hundred years, their employment in lung and liver diseases is coming on the stage only in the last few years. Placenta-derived stem cells have been recently more thoroughly characterized for their phenotype, multipotency and expression of pluripo‐ tency genes.

In CF, lung disease has been the target first of gene therapy approaches brought to the clinical stage [206, 207], hesitated in a slow progression due to limited efficiency of gene transfer vectors and pathophysiological barriers, and then of stem cell-based experimental treatments in mice [208]. Despite a very low level of engraftment of donor HSPC into the nose and the gut, significant CFTR mRNA expression and a measurable level of correction of the electro‐ physiological defect were observed after transplantation of wild-type marrow cells into CF mice [209]. It is uncertain whether this effect is due to the presence of CFTR-expressing epithelial cells derived from donor cells or to the paracrine effects of transplanted cells. Other sources, such as umbilical cord blood, embryonic stem cells, and induced pluripotent stem cells are being evaluated [210, 211]. Recent *in vitro* data on the acquisition of CFTR expression by hAMSC indicate placenta-derived stem cells as a possible source for treating the early phases of CF lung disease. Anyhow, caution should be taken when stem cell-based therapies are proposed for an inflammatory disease like that of CF lung, in view of the fact that these cells could be immunosuppressive and/or contribute to the inflammatory process. There is no available information concerning the immunomodulatory effects of placenta-derived stem cells in CF lungs.

Liver fibrosis is a common outcome of a variety of chronic liver diseases following different insults, including the biliary disorder occurring in CF. Orthotopic liver transplantation remains the only viable therapeutic option to treat CF patients with hepatic cirrhosis, and hepatocyte transplantation has never been attempted in this disease. The use of progenitor cell transplan‐ tation is emerging as a potential alternative, and several potential sources have been identified for the isolation of these cells [212]. For the treatment of liver cirrhosis, this approach has been performed mainly with BM-derived MSC [213, 214]. Given the drawbacks related to the use of BM-derived MSC (limited frequency, invasive procedure, age and disease state affecting the collection of healthy autologous BM), placenta-derived stem cells could represent a prime candidate for the treatment of liver fibrosis, since they are immunotolerated, can be isolated and produced at high yield, and do not provoke ethical debate. AM and AM-derived stem cells have been demonstrated to halt the progression of liver fibrosis and its evolution towards cirrhosis, but the long-term safety and therapeutic efficacy are not known yet, which warrant further studies. Moreover, optimal therapeutic regimens for clinical application of placentaderived stem cells, such as optimal doses, transplantation route and interval period for transplantation should be evaluated in detail [215].
