B. Linju Yen

Isolation of multipotent cells from human term placenta.

Current sources of stem cells include embryonic stem cells (ESCs) and adult stem cells (ASCs). However, concerns exist with either source: ESCs, with their significant ethical considerations, tumorigenicity, and paucity of cell lines; and ASCs, which are possibly more limited in potential. Thus, the search continues for an ethically conducive, easily accessible, and high‐yielding source of stem cells. We have isolated a population of multipotent cells from the human term placenta, a temporary organ with fetal contributions that is discarded postpartum. These placenta‐derived multipotent cells (PDMCs) exhibit many markers common to mesenchymal stem cells—including CD105/endoglin/SH‐2, SH‐3, and SH‐4—and they lack hematopoietic‐, endothelial‐, and trophoblastic‐specific cell markers. In addition, PDMCs exhibit ESC surface markers of SSEA‐4, TRA‐1‐61, and TRA‐1‐80. Adipogenic, osteogenic, and neurogenic differentiation were achieved after culturing under the appropriate conditions. PDMCs could provide an ethically uncontroversial and easily accessible source of multipotent cells for future experimental and clinical applications.

Placenta‐Derived Multipotent Cells Exhibit Immunosuppressive Properties That Are Enhanced in the Presence of Interferon‐γ

Several types of nonhematopoietic stem cells, including bone marrow mesenchymal stem cells (BMMSCs) and embryonic stem cells, have been shown to have immunosuppressive properties. We show that human placenta‐derived multipotent cells (PDMCs), which are isolated from a source without ethical concern and harbor multilineage differentiation potential, have strong immunosuppressive properties. PDMCs suppress both mitogen‐induced and allogeneic lymphocyte proliferation in both CD4 and CD8 populations. The immunosuppression seen with PDMCs was significantly stronger than that with BMMSCs. Both PDMCs and BMMSCs express indoleamine 2,3‐dioxygenase, but only PDMCs are positive for intracellular human leukocyte antigen‐G (HLA). Mechanistically, suppression of lymphocyte reactivity by PDMCs is not due to cell death but to decreased cell proliferation and increased numbers of regulatory T cells. Addition of neutralizing antibodies to interleukin‐10 and transforming growth factor (TGF)‐β partially restored lymphocyte proliferation. Unlike BMMSCs, PDMCs treated with interferon‐γ for 3 days only very minimally upregulated HLA‐DR. On the contrary, PD‐L1, a cell surface marker that plays an inhibitory role in T‐cell activation, was upregulated and TGF‐β expression was seen. The immunosuppressive properties of PDMCs, along with their multilineage differentiation potential, ease of accessibility, and abundant cell numbers, may render these cells as good potential sources for future therapeutic applications.

Brief Report—Human Embryonic Stem Cell‐Derived Mesenchymal Progenitors Possess Strong Immunosuppressive Effects Toward Natural Killer Cells as Well as T Lymphocytes

The derivation of mesenchymal progenitors from human embryonic stem cells (hESCs) has recently been reported. We studied the immune characteristics of these hESC‐derived mesenchymal progenitors (EMPs) and their interactions with T lymphocytes and natural killer cells (NKs), two populations of lymphocytes with important roles in transplantation immunology. EMPs express a number of bone marrow mesenchymal stromal cell (BMMSC) markers, as well as the hESC marker SSEA‐4. Immunologically, EMPs do not express HLA‐DR or costimulatory molecules. On the other hand, HLA‐G, a nonclassic MHC I protein involved in mediating maternal‐fetal tolerance, can be found on the surface of EMPs, and its expression is increased after interferon‐γ stimulation. EMPs can suppress CD4+ or CD8+ lymphocyte proliferation, similar to BMMSCs. However, EMPs are more resistant to NK‐mediated lysis than BMMSCs and can suppress the cytotoxic effects of activated NKs, as well as downregulating the NK‐activating receptors NKp30 and NKp46. With their broad immunosuppressive properties, EMPs may represent a new potential cell source for therapeutic use. STEM CELLS 2009;27:451–456

Synergism of biochemical and mechanical stimuli in the differentiation of human placenta-derived multipotent cells into endothelial cells.

There have been intensive studies on the differentiation of endothelial progenitor cells (EPCs) into endothelial cells. We investigated the endothelial differentiation of placenta-derived multipotent cells (PDMCs), a population of CD34(-)/CD133(-)/Flk-1(-) cells. PDMCs were cultured in basal media or media containing endothelial growth factors (EGM), including vascular endothelial growth factor (VEGF), for 3 days and then subjected to shear stress of 6 or 12dyn/cm(2) for 24h. Culture of PDMCs in EGM under static conditions resulted in significant increases in VEGF receptor-1 (Flt-1) and receptor-2 (Flk-1) expression. Application of shear stress at 12dyn/cm(2) to these cells led to significant increases in their expression of von Willebrand Factor and platelet-endothelial cell adhesion molecule-1 at both the gene and protein levels. Shear stress at 6dyn/cm(2) had lesser effects. Uptakes of acetylated low-density lipoproteins as well as formation of tube-like structures on Matrigel were significantly increased after subjecting to shear stress of 12dyn/cm(2) for 24h. Our findings suggest that the combined use of endothelial growth factors and high shear stress is synergistic for the endothelial differentiation of PDMCs.

Multipotent Human Mesenchymal Stromal Cells Mediate Expansion of Myeloid-Derived Suppressor Cells via Hepatocyte Growth Factor/c-Met and STAT3

Summary Mesenchymal stromal cells (MSCs) are multilineage progenitors with immunomodulatory properties, including expansion of immunomodulatory leukocytes such as regulatory T lymphocytes (Tregs) and tolerogenic dendritic cells. We report that human MSCs can expand CD14−CD11b+CD33+ human myeloid-derived suppressor cells (MDSCs). MSC-expanded MDSCs suppress allogeneic lymphocyte proliferation, express arginase-1 and inducible nitric oxide synthase, and increase the number of Tregs. This expansion occurs through the secretion of hepatocyte growth factor (HGF), with effects replicated by adding HGF singly and abrogated by HGF knockdown in MSCs. In wild-type mice, the liver, which secretes high levels of HGF, contains high numbers of Gr-1+CD11b+ MDSCs, and injection of HGF into mice significantly increases the number of MDSCs. Expansion of MDSCs by MSC-secreted HGF involves c-Met (its receptor) and downstream phosphorylation of STAT3, a key factor in MDSC expansion. Our data further support the strong immunomodulatory nature of MSCs and demonstrate the role of HGF, a mitogenic molecule, in the expansion of MDSCs.

Placenta-derived multipotent cells differentiate into neuronal and glial cells in vitro.

Stem cells have great potential for clinical application because of their self-renewal property and ability to differentiate into many types of cells, but because there are ethical and biological limitations with current sources of stem cells, the search continues for more suitable sources of multipotent cells. We have reported previously on a population of multipotent cells isolated from the human term placenta, an ethically unproblematic and easily available source of tissue. These placenta-derived multipotent cells (PDMCs) can differentiate into lineages of mesenchymal tissues, including osteoblasts and adipocytes, as well as non-mesenchymal tissue of neuron-like cells. We further examined the ability of PDMCs to differentiate into all 3 types of neural cells--neurons, astrocytes, and oligodendrocytes--under various induction conditions, including retinoic acid (RA), 1-methyl-3-isobutylxanthine (IBMX), and co-culture with neonatal rat brain cells. PDMCs exhibited outgrowth of processes and the expression of neuron-specific molecules such as neuron-specific enolase upon induction. Co-culture with neonatal rat brain cells also induced neural differentiation. Our results indicate that PDMCs can be differentiated into neural cell types of the human nervous system upon exposure to RA, IBMX, or primary rat brain cells.

Human mesenchymal stem cells (MSCs) for treatment towards immune- and inflammation-mediated diseases: review of current clinical trials

Human mesenchymal stem cells (MSCs) are multilineage somatic progenitor/stem cells that have been shown to possess immunomodulatory properties in recent years. Initially met with much skepticism, MSC immunomodulation has now been well reproduced across tissue sources and species to be clinically relevant. This has opened up the use of these versatile cells for application as 3rd party/allogeneic use in cell replacement/tissue regeneration, as well as for immune- and inflammation-mediated disease entities. Most surprisingly, use of MSCs for in immune-/inflammation-mediated diseases appears to yield more efficacy than for regenerative medicine, since engraftment of the exogenous cell does not appear necessary. In this review, we focus on this non-traditional clinical use of a tissue-specific stem cell, and highlight important findings and trends in this exciting area of stem cell therapy.

Multilineage Differentiation and Characterization of the Human Fetal Osteoblastic 1.19 Cell Line: A Possible In Vitro Model of Human Mesenchymal Progenitors

The in vitro study of human bone marrow mesenchymal stromal cells (BMMSCs) has largely depended on the use of primary cultures. Although these are excellent model systems, their scarcity, heterogeneity, and limited lifespan restrict their usefulness. This has led researchers to look for other sources of MSCs, and recently, such a population of progenitor/stem cells has been found in mesodermal tissues, including bone. We therefore hypothesized that a well‐studied and commercially available clonal human osteoprogenitor cell line, the fetal osteoblastic 1.19 cell line (hFOB), may have multilineage differentiation potential. We found that undifferentiated hFOB cells possess similar cell surface markers as BMMSCs and also express the embryonic stem cell‐related pluripotency gene, Oct‐4, as well as the neural progenitor marker nestin. hFOB cells can also undergo multilineage differentiation into the mesodermal lineages of chondrogenic and adipocytic cell types in addition to its predetermined pathway, the mature osteoblast. Moreover, as with BMMSCs, under neural‐inducing conditions, hFOB cells acquire a neural‐like phenotype. This human cell line has been a widely used model of normal osteoblast differentiation. Our data suggest that hFOB cells may provide for researchers an easily available, homogeneous, and consistent in vitro model for study of human mesenchymal progenitor cells.

Induction of immunomodulatory monocytes by human mesenchymal stem cell‐derived hepatocyte growth factor through ERK1/2

Monocytes are a population of leukocytes that terminally differentiate into macrophages and DCs. Whereas these differentiated progeny have inflammatory and resident—which are more immunomodulatory—phenotypes, less has been reported on the plasticity of monocytes themselves. We found that MSCs, a population of somatic stem cells, can rapidly induce human and murine monocytes through secretion of HGF to acquire an immunomodulatory phenotype to suppress T cell effector function. MSCs are multilineage postnatal progenitor cells with strong immunomodulatory effects toward T lymphocytes, NK lymphocytes, and DCs, but less is known regarding their interactions with monocytes. We found that CD14+ human monocytes express c‐Met, the receptor for HGF, and both depletion of HGF‐treated CD14+ monocytes and knockdown of HGF secretion in MSCs abrogate the suppression of anti‐CD3/28‐activated T cell proliferation. HGF‐treated monocytes remain undifferentiated and can alter activated T cell cytokine expression from a Th1 toward Th2 profile. Moreover, monocytes cocultured with MSCs or treated with HGF alone can produce high levels of IL‐10, a potent immunomodulatory cytokine. Injection of HGF to WT mice also results in an increase in IL‐10+‐expressing monocytes from the spleen, a known reservoir for circulating monocytes. Mechanistically, HGFs modulate IL‐10 production in monocytes through the ERK1/2 pathway. Our data demonstrate further the pleomorphic nature of MSC immunomodulation, as well as highlight the important role of immunomodulatory monocytes in altering T cell effector function.

Surface expression of HLA-G is involved in mediating immunomodulatory effects of placenta-derived multipotent cells (PDMCs) towards natural killer lymphocytes

Interactions between maternal natural killer lymphocytes (NKs) and fetal tissues are important in mediating maternal–fetal tolerance. We therefore investigated the interactions of NKs to placenta-derived multipotent cells (PDMCs) isolated from the term human placenta. PDMCs have similar cell surface marker expression as bone marrow mesenchymal stem cells (BMMSCs) and additionally express human embryonic stem cell markers SSEA-4 and CD-9. Differentiation into the tri-mesodermal lineages of osteoblastic, adipocytic, and chondrogenic phenotypes can be readily achieved under the appropriate conditions. We found that PDMCs are more resistant to NK-mediated lysis than the major histocompatibility complex (MHC) class-I null target cell K562, and can suppress NK secretion of interferon-γ (IFN-γ). Moreover, as third-party cells, PDMCs suppressed the cytotoxic effects of cytokine-stimulated NKs on K562. Pretreatment of PDMCs with IFN-γ, a proinflammatory cytokine, surprisingly enhanced such immunosuppressive effects. Cell–cell contact between NKs and PDMCs is required for suppressive effects, which are partially mediated by slight upregulation of the NK inhibitory receptor killer inhibitory receptor and downregulation of the activating receptor NKp30. Moreover, enhancement of PDMC suppressive effects is also mediated by IFN-γ-induced surface expression of HLA-G—an immunomodulatory nonclassical MHC class I molecule—on PDMCs, as seen by partial reversibility with HLA-G neutralizing antibodies. With its broad immunosuppressive properties, PDMCs may represent a potential cell source for therapeutic use.