Graduate School of Biomedical Sciences | Stem Cell Research and Regenerative Medicine

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PLACENTAL STEM CELLS

A Scientific Summary

Introduction
The field of stem cell research has defined a multitude of cells with plasticity of multilineage potential and self-renewal ability from almost every organ of the body, such as cardiovascular system, epidermis, gut, and even the most transient and primitive organ, the placenta. The placenta is an ephemeral organ derived from the development of the embryo and later connects the fetus to the uterine wall, by means of which the nutritive, respiratory, and excretory functions of the fetus are carried on.

Since the development of the fetus is so vigorous, the placenta may also be a reserve of various progenitor and stem cells, in addition to its function in fetal development, nutrition and immunotolerance. Placenta-derived progenitor/stem cells can be obtained without invasive procedures on embryo and does not elicit ethical debates. Because of their diversity of phenotypic plasticity and immunomodulatory properties, placenta-derived stem cells will have great future for cell therapy-based regenerative medicine.

According to the first international “Workshop on Placenta Derived Stem Cells”, which was just held in March 2007 in Italy, the research of human placenta-derived progenitor/stem cells are focused on mesenchymal stromal cells isolated from various parts of the placenta or epithelial cells isolated from amniotic membrane. These mainly four cell populations are human Amniotic Epithelial Cells (hAEC), Amniotic Mesenchymal Stromal Cells (hAMSC), Chorionic Mesenchymal Stromal Cells (hCMSC), and Chorionic Trophoblastic Cells (hCTC) (Parolini et al. 2007). Some studies have defined other names of placental progenitor/stem cells, such as Placenta-derived multipotent cells (PDMC) (Yen et al. 2005). Considering there is not uniform definition of placental stem cells until recent, some such cell populations mentioned in literatures may not actually originate from the placenta, but exist in placenta that come from maternal-fetal traffic, such as cord blood and amniotic fluid derived stem cells (here we will introduce some progress in studies of amniotic fluid-derived stem cells, umbilical cord blood would be introduced in another summary).

Basic Biology and cell development:
The placenta physically connects the mother to her developing fetus. It is responsible for providing the fetus with nutrients, oxygen, excretion; contributing to the bone development through Ca2+ ion reabsorption (Jaffredo et al. 2005); and maintaining fetal tolerance that displays immunomodulatory functions (Mellor et al. 2000). In human, the placenta has two components, the fetal part of Chorion Frondosum (chorionic plate), and the maternal part of Decidua Basailis (basal plate) (Figure 1). Fetal membrane, which consists of amnion and chorion, continues from the edge of chorionic plate and encloses fetus and amniotic fluid in amniotic cavity. In term placenta, the multilayered chorionic plate consists of two different structures (Figure 2): the amniotic membrane, which composed of amniotic epithelium (AE) and amniotic mesoderm (AM); the chorion, which composed of chorionic mesoderm (CM) and a layer of extravillous trophoblast cells. Chorionic villi originate from chorionic plate and anchor the placenta through basal plate, which is the place to separate maternal and fetus circulation and for nutrition/gas exchange. During the embryological development, the embryo, umbilical cord and amniotic epithelium are derived from the inner cell mass of blastocyst. The trophoblast of blastocyst invades into the endometrium, finally develops into different structures of placenta.

hAEC, hAMEC and hCMSC are most studied placenta-derived progenitor/stem cells. Mesenchymal stromal cells (MSC) are primarily from bone marrow (BM-MSC), to define hAMSC and hCMSC which originate from extraembroyonic mesoderm of amnion or chorion, there are five minimal criteria: 1. fetal origin (maternal contamination of 1% or less); 2. formation of fibroblast colony forming units; 3. a specific surface antigen pattern: CD45- CD34- CD14- CD90+ CD73+ CD105+ and HLA-DR-; 4. differentiation potential to one or more lineages: osteogenic, adiopogenic, chondrogenic or vascular/endothelial; 5. adherence to plastic (Parolini et al. 2007).

hAMEC and hCMSC can be isolated from first-, second- and third-trimester mesoderm of amnion and chrorion (In 't Anker et al. 2004; Wolbank et al. 2007). Both hAMSC and hCMSC are able to adhere and proliferate on tissue culture plastic, while the expression of CD49d (alpha4 integrin) distinguishes hAMSC from hAEC in cell culture (Wolbank et al. 2007). hAMSC shows both mesenchymal and epithelial ultrastrucrues but these characters are not found in hCMSC; hCMSC shows the ultrastructure of a simpler cytoplasmic organization which resembles those found in the hematopoietic progenitors (Pasquinelli et al. 2007). These studies indicate that hAMSC is multipotentiality while hCMSC is more primitive and metabolically quiescent. hAMSC and hCMSC express similar typical mesenchymal marker profiles as BM-MSC but are negative for hematopoietic markers, CD34 and CD45, and monocytic marker CD14, while they express stage specific embryonic antigens SSEA-3, SSEA-4 and OCT-4 (Alviano et al. 2007; Wolbank et al. 2007). Both hAMSC and hCMSC express low HLA-ABC and no HLA-DR, indicating their immunoprivileged status (Wolbank et al. 2007). hAMSC and hCMSC can differentiate toward classic mesodermal lineages, such as osteogenic, chondrogenic and adiopogenic lineages, for all three embryonic germ layers: ectoderm (neural); mesoderm (skeletal muscular, cardiomyocytic, endothelial); and endoderm (pancreatic) (Alviano et al. 2007; Soncini et al. 2007; Wolbank et al. 2007).

Resent studies have indicated that hAECs also express stem cell markers and have the potential to differentiate toward all three germ layers, though they are termed as epithelial cells. Cell surface markers signing stemness including CD34, CD133, and SSEA-1 are negative in hAECs; while other pluripotent stemness markers such as OCT-4, SOX-2 and Nanog are positive in hAECs (Miki et al. 2005). Studies confirmed the differentiation potential of hAEC into neural, pancreatic and hepatic cells under specific micro-environments (Parolini et al. 2007).

The detailed protocols for isolation, characterization and differentiation of hAMSC, hCMSC and hAEC are summarized excellently in tables in the report from First International Workshop on Placenta Derived Stem Cells, please refer to the report (Parolini et al. 2007).

PDMCs are characterized as CD34- and CD45- cells derived from the placenta. They express CD105/endoglin/ and SH-2, SH-3, SH-4 that are common markers found on MSCs, but lack hematopoietic-, endothelial-, and trophoblastic-specific cell markers. PDMCs have a fibroblastoid morphology and plastic-adherence nature further resembling MSCs. They also express embryonic stem cell (ESC) specific and embryonic germ cell (EGC) specific markers, such as SSEA-4, TRA-1-60, TRA-1-81. This suggests that PDMCs not only have properties of MSCs, but also ESCs and EGCs (Yen et al. 2005).

The usage of amniotic fluid cells in routine genetic prenatal diagnosis has been widely and well established; however the existence of a human amniotic fluid stem cell (AFS) type was not suggested untill 2004 (Tsai et al. 2004). A distinct population of OCT-4 positive cells can be found in the background of OCT-4- amniotic fluid cells. These cells also express CD44, CD105 and HLA-ABC that resemble mesenchymal markers, but lack hematopoietic markers such as CD34 and CD45. Recently for the first time it is reported that AFS cells can be induced into six distinct lineages (adipogenic, osteogenic, myogenic, endothelial, nerogenic, and hepatic) of all three germ layers (De Coppi et al. 2007). AFS cells are an intermediate stage between ESCs and lineage-restricted adult progenitor cells. To date, further basic research of AFS cells is essential and clinical studies must be initiated.

Implications for Medicine and Applications for Diseases Therapy:
The use of MSCs is limiting in medicine because rates of infection are high during cell aspiration from the bone marrow, the procedure is invasive, and there is a lower number of available MSCs in elderly patients. ESCs have thus far been considered the best source for pluripotent cells, since they can form cells of every lineage and can be considered for cell-based therapy. However, ethical concerns regarding the destruction of the embryo form the basis for non-approval if federal funds are used for the research. ESCs are also known to cause teratomas and can also be rejected by unmatched host. Placenta-derived progenitor/stem cells have attracted a lot of attention as a cell source for cell-transplantation and regeneration therapy, because of their considerable advantageous characteristics: 1. Pluripotency and plasticity to differentiate into all three embryonic germ layers. 2. Immunoprivileged status and anti-inflammatory functions. 3. Relative non-tumorigenicity. 4. Little ethical issues with usage. The recent studies have been reported on the potential applications of placenta stem cells for regeneration of hepatocytes, cardiomyocytes, chondrocytes, insulin-producing pancreatic β-cells or neuronal cells; for repairing of auditory system or ophthalmology; and for tissue engineering (Figure 3).

Also the mechanisms of Maternal acceptance of the fetal as allograft is not so clear till today, many studies have been done and indicated that fetal membranes from placenta have immunoregulatory properties which lead to key function in maternal-fetus immune tolerance. Amniotic membrane and hAECs are been used in wounds healing of skin, burn, chronic leg ulcers and surgical procedures for decades of years. Recent in vivo study showed that amniotic membrane, which was used in ocular surface reconstruction of corneal or conjunctival epithelium, particularly had no immunosuppression or acute rejection responses (Gomes et al. 2005). In vitro studies also revealed that cells isolated from amniotic and chorionic membranes actively suppress lymphocyte proliferation without eliciting allogeneic or xenogeneic immune responses (Bailo et al. 2004; Li et al. 2005).

In animal model study, mouse placenta is considered as potent resource and functional niche for hematopoietic stem cells (HSC). Allantois and chorion isolated in the conceptus express Runx1 (a key transcriptional factor for HSC ontogeny) and CD41 (a hallmark for initiation of hematopoiesis), and also generate myeloid and erythroid lineages following explant culture (Corbel et al. 2007; Zeigler et al. 2006), indicating placenta stem cells in usage of regeneration of hematopoietic system. It has been reported that hAMSC cells differentiate into hepatocyte-like cells in vitro (Tamagawa et al. 2007). The mixed ester of Hyaluronan, Butyric and Retinoic Acid was reported to drive cardiogenic and vasculogenic differentiation in human amniochorionic-derived cells, and such hAMSCs transplantation enhanced cardiac repair in infracted rat hearts (Ventura et al. 2007). The transplantation of hAECs improved hindlimb functions in rats with spinal cord injury (Wu et al. 2006). These studies indicated the application of placenta-derived stem cells in hepatic regeneration, cardiac repair and the potential treatments of neurological disorders.

PDMCs have been reported to suppress the functions of the proinflammatory cytokines such as IFN-γ and TGF-β action, suggesting their immunosuppressive properties as good potential for therapeutic applications (Chang et al. 2006). PDMCs also have been successfully induced into hepatocyte-like cells (Chien et al. 2006) and insulin-positive cells (Chang et al. 2007).

The usage of amniotic membrane has had a history of a hundred year, the technology of placental tissue banking has been well established, while the experience of cell banking of placenta stem cells has been gained from cord blood banking. In summary, since this decade, from basic research to preclinical studies, placenta has been holding much promise for the development of cell based therapies for clinical applications in the near future.

Figure 1: The structure of placenta (edit from figure in reference: (Parolini et al. 2007) )

Figure 2: Histology H&E slide representing cross-section of fetal part of placenta and fetal membrane (edit from figure in reference: (Parolini et al. 2007) )

Figure 3: Potential applications of placental stem cells in cell-based therapy of regenerative medicine.

REFERENCES
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Summarized by students in the Graduate Course in Stem Cell Biology: Xiangwen Chen, Laura Ellen Mahoney and MaryAnn Murillo (In alphabetical order)
Teaching Assistant: Reema Patel