Characterization of several placenta-derived stem cells. (a) Morphology of amnion epithelial cells (AEC), chorionic plate-derived mesenchymal stem cells (CP), villi-derived mesenchymal stem cells (CV), and umbilical cord-derived stem cells (UC). Scale bars = 50 μm. (b) Doubling time analysis of third to eleventh passage placenta-derived stem cells. (c) RT-PCR for self-renewal markers (Oct-4, Nanog, Sox-2 and TERT), three germinal layer markers (NF-68, Cadiac, AFP) and immunomodulatory marker (HLA-G). β-actin was used as internal control. (d) Immunophenotype of placenta-derived stem cells. Third passage CP-MSCs and UCMSCs were positive for CD90, CD44, CD13, and CD105 and negative for CD45, CD34, and CD31 by analysis of flow cytometry. It shows the data of HLA-ABC, HLA-DR, and HLA-G. Positive cells were evaluated compared with signal of isotype control and the percentages were indicated along with the fluorescence intensities. (e) Differentiation potentials of placenta-derived stem cells into mesodermal lineages including adipogenic (Oil-red O staining, upper), osteogenic (von Kossa staining, middle) and chondrogenic (Alcian Blue staining, lower) lineages. The cells were counterstained with hematoxylin. Scale bars = 50 μm. (Adapted from )
Besides the regenerative function, another important potential of placenta-derived MSCs is immune-related property [9, 52, 53]. In our data, placenta-derived MSCs were positive for HLA-ABC and negative for HLA-DR, which are similarly expressed in BM-MSCs and AD-MSCs, however, HLA-G was strongly positive only in CP-MSCs in contrast to BM-MSCs and AD-MSCs, consistent with previous report  (Fig. 14.2). In a number of in vitro studies, immunosuppressive effects of MSCs and their mechanism have been well described in which suppression of T-cell proliferation and inhibition of dendritic cell differentiation have been suggested as the key events. As well as, placenta-derived MSCs suppress allogeneic T-cell proliferation through 2,3-dioxygenase (IDO) induction secreted by IFN-r [9, 54]. Immunosuppressive effects have been further confirmed in in vivo studies and are being evaluated in clinical trials in diseases such as refractory graft-versus host-disease and Crohn’s disease. In addition, immune privilege and hypoimmunogenicity of placenta-derived MSCs are other aspects of immune-related property. Although controversy exists, there are evidences that MSC might be immune privileged to freshly isolated NK cells preventing them from lysis and that low expression of human leukocyte antigen (HLA) major histocompatibility complex (MHC) class I with no expression of co-stimulatory molecules may play an important role. These properties are anticipated to give advantage to survival and engraftment of MSCs in transplanted setting [55, 56].
Immunomodulatory effects of MSCs derived from placenta, bone marrow, and adipose on activated T-cells depend on co-cultured MSCs in dose-dependent manner. (a) MNCs isolated from umbilical cord blood were cultured with or without 1 μg/mL anti-CD3 and anti-CD28 for 72 h, which are T-cell activating mAbs, and clustering of activated T-cells decreases with increasing number of co-cultured MSCs. Scale bar = 50 μm. (b) Expression of surface markers for immunomodulation in MSCs derived from placenta, bone marrow, and adipose using flow cytometry analysis. Notably, the expression of HLA-G, which plays a role in immune tolerance, was highly positive in CP-MSCs. The percentages are indicated along with the fluorescence intensities. MSCs mesenchymal stem cells, HLA human leukocyte antigen, CP-MSCs chorionic plate-derived MSCs, BM-MSCs bone marrow-derived MSCs, AD-MSCs adipose-derived MSCs, WI-38 normal fibroblast cell line (Adapted from )
1.2 Part II. Therapeutic Potentials of PDSCs in Hepatic Diseases
Transplantation of hepatocytes has been suggested as a useful therapeutic approach for liver disease . However, there are several stumbling blocks to the use of hepatocytes in cell-based clinical applications, including difficulty of access and low yield [58, 59]. Due to the reason, many scientists have been reported and developed that the potentials on hepatogenic differentiation of MSCs derived from bone marrow, umbilical cord and adipose, and their efficacies when they transplanted into animal model with hepatic diseases such as liver fibrosis and cirrhosis, even hepatocellular carcinoma [22, 60–62].
Placenta-derived MSCs originated from mesodermal lineages also have the potential to differentiate in vitro into hepatocyte-like cells and insulin-positive cells, which are endodermal lineages [12, 13, 22]. In previous reports, isolated various types of stem cells from placental tissues including amniotic fluid, AECs, chorionic plates, villi, and umbilical cord have been characterized the stem cell capacities and hepatogenic differentiation potentials. Especially, human amniotic membrane-derived mesenchymal stem cells (hAMCs) and chorionic plate-derived mesenchymal stem cells (CP-MSCs) have been reported that their capacity to differentiate into hepatocyte-like cells, as well as their ability to reduce chronic fibrogenesis [22, 63, 64]. Also, Jung et al. reported that human umbilical cord blood (UCB)-derived MSCs could improve liver fibrosis in rat with carbon tetrachloride (CCl4)-induced cirrhosis . However, these results are controversial, as they vary somewhat among different animal models, CCl4 treatment conditions, and cell conditions [62, 66]. So, it is necessary to establish animal model with hepatic diseases to validate the therapeutic effect of PDSCs on hepatic failures. Generally, CCl4 is widely used experimentally to elicit liver damage; however, it decomposes in the lipids of damaged cells and leads to rapid breakdown of the endoplasmic reticulum and loss of its function. CCl4-induced liver injury is both severe and extremely rapid in its onset, and it ultimately manifests histologically as hepatic steatosis, fibrosis, hepatocellular death, and carcinogenicity, Nevertheless, damaged hepatocytes regenerate easily in the absence of further exposure to CCl4 . Animal injuries caused by low-dose and short-term injection of CCl4 show rapid hepatocyte regeneration on a time scale dependent on the species used, making it necessary to confirm liver damage comparable with cirrhosis in any CCl4-injured liver model [68, 69]. Therefore, many scientists utilize a rat model of severe liver injury such as early stage of cirrhosis through high-dose, long-term treatment with CCl4, or late stage of hepatic failure model by bile duct ligation (BDL) model [22, 70, 71].
Until now, several hypotheses for therapeutic mechanism of PDSCs on hepatic diseases have been introduced [22, 64, 72–74], however, it is still unclear. The therapeutic mechanism could be categorized as a cell-to-cell interaction and paracrine effect of PDSCs transplantation [75, 76]. Growing studies have explained two important aspects of MSC for cell therapy: (1) MSCs can modulate immunological responses, and (2) systemically administered MSCs home to sites of injury. Fetal-origin cells have a nature of migration ability cross the placental and blood–brain barriers. Due to their characterization, PDSCs also observed to home to the damaged liver after intravenous, portal vein and intrasplenic transplantation [77, 78]. The effect of engrafted PDSCs has been shown to increase depending on their transplantation . So, it is important factor that transplantation technology of the cells via an appropriate route is the key to maximizing the therapeutic effects of PDSCs based therapies in liver injury. Because of the effect on the homing efficiency and biological activity of stem cells, route of transplantation is one of the most important factors determining therapeutic efficacy. In our previous report, we further investigated whether different transplantation routes, namely direct transplantation into the liver (DTP), intrasplenic transplantation (STP) and intravenous transplantation via the tail vein (TTP), exhibited different therapeutic effects and which is the optimal transplantation technique for the carbon tetrachloride (CCl4)-injured liver rat model . Homing to liver tissues damaged was confirmed by tracing cells labeled with PKH26. The differences for these three different transplantation routes can be explained as follows: (1) DTP may cause loss of the fewest transplanted cells, with many cells being expected to settle in damaged liver tissues; however, surgical procedures are unavoidable and direct injury to the liver cannot be ruled out, (2) STP is known to be an effective method for cell transplantation, with efficient homing of cells to the liver , and does not cause direct surgical damage to the liver, and (3) TTP may result in the loss of large numbers of transplanted cells during homing to damaged liver tissues, but is relatively easy and does not involve surgical procedures. Although it was revealed that DTP and STP were superior to TTP on anti-fibrotic effects, direct surgical application of stem cells to an already injured organ may exacerbate the damage. Therefore, STP would be desirable to transplant the cells by a similarly effective method that is potentially less damaging to the target organ. It was found that liver regeneration was more greatly enhanced in the STP group than in the DTP group although the CCl4 administration cause injury of spleen called megalosplenia as well as liver. The therapeutic effects of engrafted PDSCs were systematically investigated by comparing DTP, STP, and TTP. Based on these findings, STP may represent an appropriate method for administering cell-based therapies in the treatment of liver disease.
The major therapeutic effect of PDSCs on a rat model with fibrosis or cirrhosis is an anti-fibrotic effect through increased MMP-2 and MMP-9 activities [22, 72, 74, 75, 78] (Fig. 14.3). Engrafted PDSCs inhibit collagen synthesis, trigger the expression of MMPs, and modulate MMP activities in TGF-β-exposed T-HSC/Cl-6 cells in an in vitro co-culture system . These mechanisms are similar to those of previous reports, which have shown that transplanted bone marrow-derived mesenchymal stem cells (BM-MSCs) increase MMP expression in liver failure models [82, 83]. Manuelpillai et al. reported that human amnion epithelial cells (hAECs) engrafted onto immunocompetent CCl4-injured mice show lower expression of TNF-a and IL-6, and higher IL-10 comparing to mice model without transplantation of hAECs. Also, engrafted hAEC induced to decrease the expression of hepatic TGF-b levels, which is capable of controlling the expression of the collagen through regulation of degrading enzyme (e.g., MMP-2) . These histopathological improvements in animal model transplanted with PDSCs may be linked to the observed improvement in function of the damaged liver. Furthermore, Cargnoni et al. reported that transplantation of allogenic and xenogenic PDSCs reduced both neutrophil infiltration and fibrosis in mice with bleomycin-induced lung fibrosis within 2 weeks . These reports suggest the possibility that PDSCs may be useful therapeutic reagents in the treatment of degenerative diseases characterized by abnormal collagen deposition.
Therapeutic effects of the transplantation of placenta-derived stem cells into the CCl4-injured rat liver. Liver tissues were obtained from TP group (Upper) and Non-TP group (Lower) at 1, 2 and 3 weeks after generation of the CCl4-injured rat liver disease model for 9 weeks. A: After CP-MSCs transplantation into liver tissues, the degree of liver fibrosis was analyzed from 1 week to 3 weeks using MT staining. (×100). (Adapted from )
Also, PDSCs have immunomodulatory properties. The utility of placenta-derived stem cells is being explored towards wider range of areas and is incorporated in clinical trials as well based on the reports of their superior immunomodulatory effects. Indeed, considering the fundamental role of placenta as a bridge between mother and fetus, the immunomodulatory function might be inherited by PDSCs [35, 85]. Jung et al. demonstrated the transplantation of PDSCs into the injured rat liver resulted in a dramatic reduction of leukocytic infiltration. Additionally, IL-10 expression was significantly increased compared to the NTP group at 1 week and 3 weeks post transplantation (Fig. 14.4). These findings suggest that transplantation of PDSCs improves injured liver tissue conditions, at least in part through anti-inflammation processes . The results are similar to previous reports [9, 51, 78, 86]. The anti-inflammatory effects of PDSCs suppress the chronic inflammation condition of hepatic diseases resulting in an improved environmental condition to recover liver damage.
Anti-inflammatory effect on rat injured liver according to placenta-derived stem cells transplantation. Quantitative ELISA analysis of the anti-inflammatory cytokine IL-10. Equal amounts of protein from individual animals were pooled from control rats (n = 5), TP 1 week (n = 6), TP 2 weeks (n = 6), TP 3 weeks (n = 7), NTP 1 week (n = 6), NTP 2 weeks (n = 6), NTP 3 weeks (n = 7) and analyzed by ELISA. All reactions were performed in triplicate. Data are expressed as the mean ± SD of triplicate experiments. Significant differences were observed at 1 week and 3 weeks post transplantation (*p < 0.05). CTL control rat, TP CP-MSCs-transplanted group, NTP non-transplanted group, wk. week(s) (Adapted from )
The balance between cell death and survival is critical for several cellular processes, including embryogenesis, organogenesis, repair systems and carcinogenesis and tissue and organ repair consists of complex, multicellular processes involving the coordinated efforts of inflammation, survival and/or cell death regulation [87, 88]. Fujiyoshi et al. reported that, in liver regeneration, cell proliferation and cell growth are achieved through interleukin (IL)-6/STAT3 and PI3K/PDK1/Akt pathways via downstream molecules, such as cyclin D1/p21 and mammalian target of rapamycin (mTOR) expression, and protect against cell death by upregulating anti-apoptotic factors (e.g., FLIP, Bcl-2, Bcl-xL, Ref1 and MnSOD) . Recently, it was reported that proliferation or apoptosis mechanisms are regulated by autophagy, which is considered a key-regulating factor of disease-repair processes [90, 91]. Jung et al. demonstrated that placenta-derived mesenchymal stem cells promote hepatic regeneration in CCl4-injured rat liver via through upregulation of autophagy-inducing factors (e.g., PI3K class III, Beclin1, ATG7, ATG5-12 and LC3 II) as well as downregulation of negative regulator (e.g., p-mTOR) of autophagy  (Fig. 14.5). This finding suggests a new therapeutic mechanism that PDSCs transplantation induces hepatic cell regeneration in injured livers via upregulation of autophagy-related signaling molecules.
Expression levels of cell cycle-, liver regeneration- and tissue regeneration-related factors in liver tissues from the TP and NTP groups. (a) The proliferative activity of liver tissues from rats in the control, TP, and NTP groups assessed through immunohistochemical analysis of Ki67 and the Ki67 labeling index (Ki-LI) and presented as the percentage of Ki67-positive nuclei in the total number of hepatocytes. Data expressed as mean ± SD. *Depicts significant differences between the TP and NTP groups (p < 0.001). (b) Caspase 3/7 activities from liver tissues of the TP and NTP groups. The activities of caspase 3/7 by ELISA in the liver tissues of the TP and NTP groups. Equal amounts of protein from individual animals were pooled from control rats (CTL; n = 5), TP 1 week (n = 6), TP 2 weeks (n = 6), TP 3 weeks (n = 7), NTP 1 week (n = 6), NTP 2 weeks (n = 6), NTP 3 weeks (n = 7) and loaded each well of ELISA kit. All reactions were performed in triplicate. Data are expressed as the mean ± SD of triplicate experiments. *Depicts significant differences between the TP and NTP groups (p < 0.05). (c) The expression of total- (t-), phosphorylated- (p-) mTOR, PI3K III, Beclin1, ATG7, ATG5-12, LC3 I, and LC3 II in the liver tissues of the TP and NTP groups. Actin was used as a loading control. (Adapted from )
Stem cell-based therapy represents a novel approach for regenerative medicine and treatment of a wide spectrum of disorders affecting diverse organ systems. Being derived from the placenta, PDSCs (e.g., amnion, chorion, umbilical cord, and Wharton’s jelly) have immunomodulatory properties, suggesting that the transplantation of PDSCs may reduce inflammatory responses in injured organs [44, 54]. Recently, Ichim and their colleagues reported that a clinical trial with placenta-derived allogenic mesenchymal stem cell treatment in patients with symptoms of either acute or chronic graft-versus host-disease was well tolerated and no adverse side effects were observed either acutely or 11 months posttreatment period . These properties of PDSCs are considered very strong advantages for cell therapy through allogenic transplantation for stable engraftment via escape from host immune rejection. Furthermore, PDSCs have been added to the growing list of putative stem cell populations with providing some advantages such as easily accessibility from the tissues generally discarded after birth, harvest a lot of early stage of several distinguishable adult stem cells as well as overcomes ethical concerns. Also, PDSCs grow well in culture and appear capable of differentiation to multiple cell types including hepatocyte-like cells. The therapeutic potential of PDSCs have been demonstrated several degenerative diseases (e.g., neuronal-, skeletal disease, diabetes, as well as liver disease) by differentiation into the functional cells.
Many scientists have been demonstrated that PDSCs have the therapeutic potential in hepatic diseases. Based on the above studies, we propose that the repair mechanism triggered by PDSCs transplantation in injured liver acts through multiple events as follows: (1) environmental rehabilitation through anti-fibrosis and anti-inflammation, (2) damaged cells clearance through apoptosis and autophagy, (3) cellular protection and recycling of cellular products through autophagy, and (4) functional cells repopulation through activation of cell proliferation in injured hepatic cells (Fig. 14.6). PDSCs are an attractive cell source for degenerative diseases including hepatic diseases.
Therapeutic mechanism of placenta-derived stem cells in hepatic diseases. The repair mechanism triggered by placenta-derived stem cell transplantation in injured liver acts through multiple events as follows: (1) environmental rehabilitation through anti-fibrosis and anti-inflammation, (2) damaged cells clearance through apoptosis and autophagy, (3) cellular protection and recycling of cellular products through autophagy, (4) functional cells repopulation through activation of cell proliferation in injured hepatic cells
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