Antifibrotic Activity of Human Placental Amnion Membrane-Derived CD34+ Mesenchymal Stem/Progenitor Cell Transplantation in Mice With Thioacetamide-Induced Liver Injury.

: Liver fibrosis represents the end stage of chronic liver inflammatory diseases and is defined by the abnormal accumulation of extracellular matrix in the liver. Advanced liver fibrosis results in cirrhosis, liver failure, and portal hypertension. Liver transplantation has been the most effective treatment for these diseases, but the procedure is limited by the shortage of suitable donors. Mesenchymal stromal cells (MSCs) have shown great potential in the treatment of chronic inflammatory diseases associated with fibrosis. This study aimed to evaluate the therapeutic effect of MSC-based cell transplantation as an alternative treatment for liver fibrosis. A CD34-positive subpopulation of human placental amnion membrane-derived stem/progenitor cells (CD34+ AMSPCs) was isolated through the depletion of CD34-negative stromal fibroblasts (CD34- AMSFCs) facilitated by CD34 fluorescence-activated cell sorting, enriched and expanded ex vivo. These cells express pluripotency markers and demonstrate multidirectional differentiation potentials. Comparative analysis was made between CD34+ AMSPCs and CD34- AMSFCs in terms of the expressions of stemness surface markers, embryonic surface antigens, and multilineage differentiation potentials. A mouse model of liver fibrosis was established by thioacetamide (TAA) administration. When injected into the spleen of TAA-injured mice, human placental amnion membrane-derived MSCs (hAM-MSCs) can engraft into the injury site, ameliorate liver fibrosis, and restore liver function, as shown by pathological and blood biochemical analysis and downregulated gene expressions associated with liver damage. CD34+ AMSPCs represent a more primitive subset of hAM-MSCs and could be a suitable candidate with a potentially better safety profile for cell-based therapy in treatment of liver diseases associated with fibrosis. SIGNIFICANCE: In this study, a CD34+ subpopulation of stem/progenitor cells derived from neonatal placental amnion membrane, denoted as CD34+ AMSPCs, were identified, enriched, and characterized. These cells are highly proliferative, express mesenchymal stromal cells and pluripotent stem cell markers, and demonstrate multidirectional differentiation potentials, indicating their promising application in clinical regenerative therapies. CD34+ AMSPC transplantation ameliorated liver fibrosis in mice with drug-induced liver injury. These cells represent a potential therapeutic agent for treating liver diseases associated with fibrosis.

Gene methylation of human ovarian carcinoma stromal progenitor cells promotes tumorigenesis.

BACKGROUND: This study aimed to investigate whether the DNA methylation of human ovarian carcinoma stromal progenitor cells (OCSPCs) could promote the tumorigenesis of ovarian carcinoma. METHODS: OCSPCs were first isolated from fresh tumor tissues and ascites of ovarian cancer patients. In vivo and in vitro experiments on the effect of the OCSPCs on tumorigenesis and the effects of DNA demethylation on the OCSPCs were then performed. RESULTS: The OCSPCs possessed self-renewal and multipotent differentiation capacity with elevated expressions of OCT4, NANOG, BMP2, BMP4, Rex-1, AC133 and TGF-ß. The OCSPCs, when combined with tumor cells in vivo could promote tumor growth. The methylation profiles of tumor suppressor genes (TSGs) were significantly higher in the OCSPCs than in ovarian cancer cells (p < 0.001). 5-aza-2-dC could alter the methylation levels of TSGs in OCSPCs and also inhibit the tumor promoting capabilities of the OCSPCs by decreasing the proliferation of tumors cells. The expression levels of TSGs were re-expressed by 5-aza-2-dC to inhibit the self-renewal and growth of OCSPCs. CONCLUSIONS: OCSPCs with decreased TSG expressions in the ovarian tumor microenvironment were able to promote tumorigenesis which could be reversed by DNA demethylation. DNA demethylation reversing the expression of TSGs in OCSPCs may represent a potential therapeutic target for ovarian cancer.

Preparation, quality criteria, and properties of human blood platelet lysate supplements for ex vivo stem cell expansion.

Most clinical applications of human multipotent mesenchymal stromal cells (MSCs) for cell therapy, tissue engineering, regenerative medicine, and treatment of immune and inflammatory diseases require a phase of isolation and ex vivo expansion allowing a clinically meaningful cell number to be reached. Conditions used for cell isolation and expansion should meet strict quality and safety requirements. This is particularly true for the growth medium used for MSC isolation and expansion. Basal growth media used for MSC expansion are supplemented with multiple nutrients and growth factors. Fetal bovine serum (FBS) has long been the gold standard medium supplement for laboratory-scale MSC culture. However, FBS has a poorly characterized composition and poses risk factors, as it may be a source of xenogenic antigens and zoonotic infections. FBS has therefore become undesirable as a growth medium supplement for isolating and expanding MSCs for human therapy protocols. In recent years, human blood materials, and most particularly lysates and releasates of platelet concentrates have emerged as efficient medium supplements for isolating and expanding MSCs from various origins. This review analyzes the advantages and limits of using human platelet materials as medium supplements for MSC isolation and expansion. We present the modes of production of allogeneic and autologous platelet concentrates, measures taken to ensure optimal pathogen safety profiles, and methods of preparing PLs for MSC expansion. We also discuss the supply of such blood preparations. Produced under optimal conditions of standardization and safety, human platelet materials can become the future 'gold standard' supplement for ex vivo production of MSCs for translational medicine and cell therapy applications.

Isolation and characterization of stromal progenitor cells from ascites of patients with epithelial ovarian adenocarcinoma.

BACKGROUND: At least one-third of epithelial ovarian cancers are associated with the development of ascites containing heterogeneous cell populations, including tumor cells, inflammatory cells, and stromal elements. The components of ascites and their effects on the tumor cell microenvironment remain poorly understood. This study aimed to isolate and characterize stromal progenitor cells from the ascites of patients with epithelial ovarian adenocarcinoma (EOA). METHODS: Seventeen ascitic fluid samples and 7 fresh tissue samples were collected from 16 patients with EOA. The ascites samples were then cultured in vitro in varying conditions. Flow cytometry and immunocytochemistry were used to isolate and characterize 2 cell populations with different morphologies (epithelial type and mesenchymal type) deriving from the ascites samples. The in vitro cell culture model was established using conditional culture medium. RESULTS: The doubling times of the epithelial type and mesenchymal type cells were 36 h and 48 h, respectively, indicating faster growth of the epithelial type cells compared to the mesenchymal type cells. Cultured in vitro, these ascitic cells displayed the potential for self-renewal and long-term proliferation, and expressed the typical cancer stem/progenitor cell markers CD44(high), CD24(low), and AC133(+). These cells also demonstrated high BMP-2, BMP4, TGF-ß, Rex-1, and AC133 early gene expression, and expressed EGFR, integrin α2ß1, CD146, and Flt-4, which are highly associated with tumorigenesis and metastasis. The epithelial type cells demonstrated higher cytokeratin 18 and E-cadherin expression than the mesenchymal type cells. The mesenchymal type cells, in contrast, demonstrated higher AC133, CD73, CD105, CD117, EGFR, integrin α2ß1, and CD146 surface marker expression than the epithelial type cells. CONCLUSION: The established culture system provides an in vitro model for the selection of drugs that target cancer-associated stromal progenitor cells, and for the development of ovarian cancer treatments.

Downregulation of alpha-fetoprotein expression by LHX4: a critical role in hepatocarcinogenesis.

LHX4 is a member of the LIM-homeobox family and plays a critical role in pituitary development and differentiation. Several lines of evidences have reported their aberrant expression in cancers. However, the exact roles of LHX4 in carcinogenesis remain unclear. In this study, LHX4 expression was analyzed in tumor and paired non-tumor tissues obtained from patients with hepatocellular carcinoma (HCC) using western blotting and immunohistochemistry. LHX4 was found to be downregulated in tumor tissues and negatively correlated with differentiation grade and alpha-fetoprotein (AFP) levels in 66 HCC patients. To clarify the biological functions of LHX4, transient or stable transfectants overexpressing LHX4 were generated in human hepatoma cells (Huh7 and HepG2). LHX4 overexpression in Huh7 and HepG2 cells induced a more differentiated phenotype by reducing AFP expression. Using in silico analysis, the evolutionary conserved region within the AFP promoter containing LHX4-binding site was identified, implying that AFP is a putative target for LHX4. Moreover, ectopic LHX4 overexpression attenuated Huh7 and HepG2 proliferation. Importantly, the growth-inhibitory effect of LHX4 was reversed by replenishing AFP to the LHX4-overexpressing cells, providing a functional relevance between LHX4 and AFP. Finally, we analyzed expressions of LHX4 and AFP during normal liver development. Hepatic LHX4 expression increased in adult liver in a manner that parallel AFP repression. In conclusion, these data indicate that LHX4 may act as a potential tumor suppressor in hepatocarcinogenesis, suggesting that targeting LHX4 to downregulate AFP might have therapeutic implications.

Expansion of adipose tissue mesenchymal stromal progenitors in serum-free medium supplemented with virally inactivated allogeneic human platelet lysate.

BACKGROUND: Single-donor or pooled platelet lysates (PL) can substitute for fetal bovine serum (FBS) for mesenchymal stromal cell (MSC) expansion. However, for clinical applications of MSCs, the use of virally inactivated PL would be desirable. Recently, we have developed a solvent/detergent (S/D)-treated human PL preparation (S/D-PL) rich in growth factors. The capacity to use this virally inactivated preparation for MSC expansion needs to be evaluated. STUDY DESIGN AND METHODS: Platelet concentrates were treated by S/D (1% tri-n-butyl phosphate and 1% Triton X-45), extracted by oil, purified by C18 hydrophobic interaction chromatography, and sterile filtered. S/D-PL was compared to FBS as a medium supplement (10% vol/vol) for isolating, maintaining, and expanding adipose tissue-derived MSCs (AT-MSCs). Cell morphology; proliferation kinetics; immunophenotype; differentiation capacity toward the chondrogenic, osteogenic, and osteogenic lineages; and cytokine antibody array were assessed. RESULTS: AT-MSCs had a typical spindle morphology and proliferated in S/D-PL at least as well as in FBS. Immunophenotype at Passage 7 was characteristic of MSCs and similar for both culture conditions. Differentiation capacity into the three lineages was maintained and chondrogenesis was enhanced by S/D-PL. In a 120 human cytokine antibody array analysis, 73 cytokines were detected in S/D-PL, including 22 with a concentration higher than in FBS. CONCLUSION: S/D-PL is an alternative to FBS for AT-MSC maintenance and expansion, does not compromise the differentiation capacity nor the immunophenotype, and may accelerate chondrogenesis. S/D-PL protocols for MSC clinical scale-up may represent a major step toward challenging new use in stem cell therapies.

Effect of Reishi polysaccharides on human stem/progenitor cells.

The polysaccharide fraction of Ganoderma lucidum (F3) was found to benefit our health in many ways by influencing the activity of tissue stem/progenitor cells. In this study, F3 was found to promote the adipose tissue MSCs' aggregation and chondrosphere formation, with the increase of CAM (N-CAM, I-CAM) expressions and autokine (BMP-2, IL-11, and aggrecan) secretions, in an in vitro chondrogenesis assay. In a stem cell expansion culture, it possesses the thrombopoietin (TPO) and GM-CSF like functions to enhance the survival/renewal abilities of primitive hematopoietic stem/progenitor cells (HSCs). F3 was found to promote the dendrite growth of blood mononuclear cells (MNCs) and the expression of cell adhesion molecules in the formation of immature dendritic cells (DC). On the other hand, F3 exhibited inhibitory effects on blood endothelial progenitor (EPC) colony formation, with concomitant reduction of cell surface endoglin (CD105) and vascular endothelial growth factor receptor-3 (VEGFR-3) marker expressions, in the presence of angiogenic factors. A further cytokine array analysis revealed that F3 indeed inhibited the angiogenin synthesis and enhanced IL-1, MCP-1, MIP-1, RANTES, and GRO productions in the blood EPC derivation culture. Collectively, we have demonstrated that the polysaccharide fraction of G. lucidum F3 exhibits cytokine and chemokine like functions which are beneficial to human tissue stem/progenitor cells by modulating their CAM expressions and biological activities. These findings provide us a better the observation that F3 glycopolysaccharides indeed possesses anti-angiogenic and immune-modulating functions and promotes hematopoietic stem/progenitor cell homing for better human tissue protection, reducing disease progression and health.

Disparate mesenchyme-lineage tendencies in mesenchymal stem cells from human bone marrow and umbilical cord blood.

Bone marrow and umbilical cord blood are reported to be the main sources of mesenchymal stem cells (MSCs), which have been proposed for many clinical applications. This study evaluated and quantitated the differentiation potential of bone marrow-derived MSCs (bmMSCs) and cord blood-derived MSCs (cbMSCs) by in vitro induction. Results indicated that cbMSCs had a significantly stronger osteogenic potential but lower capacity for adipogenic differentiation than bmMSCs. Leptin, an important regulator of mesenchymal differentiation, has a significantly stronger effect of promoting osteogenesis and inhibiting adipogenesis in bmMSCs than in cbMSCs. Moreover, Cbfa1 mRNA expression in bmMSCs and cbMSCs was affected to different degrees by leptin during osteogenesis. In contrast, leptin reduced PPARgamma2 mRNA expression to the same level during adipogenesis in both types of MSCs. These results demonstrate the disparate capacities of MSCs from bone marrow and cord blood and suggest that they be used differently in experimental and therapeutic studies. In addition, the disparate differentiation tendencies of MSCs from different sources should be considered in further applications.

Mesenchymal stem cells are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction.

Both cell therapy and angiogenic growth factor gene therapy have been applied to animal studies and clinical trials. Little is known about the direct comparison between cell therapy and angiogenic growth factor gene therapy. The goal of this study was to compare the effects of human bone marrow-derived mesenchymal stem cells (hMSCs) transplantation and injection of angiogenic growth factor genes in a model of acute myocardial infarction in mice. The hMSCs were obtained from adult human bone marrow and expanded in vitro. The purity and characteristics of hMSCs were identified by flow cytometry and immunophenotyping. Immediately after ligation of the left anterior descending coronary artery in male severe combined immunodeficient (SCID) mice, culture-expanded hMSCs or angiogenic growth factor genes were injected intramuscularly at the left anterior free wall. The engrafted hMSCs were positive for cardiac marker, desmin. Infarct size was significantly smaller in the hMSCs-treated group than in the angiopoietin-1 (Ang-1) or vascular endothelial growth factor (VEGF)-treated group at day 28 after infarction. hMSCs transplantation was better in decreasing left ventricular end-diastolic dimension and increasing fractional shortening than Ang1 or VEGF gene therapy. Capillary density was markedly increased after hMSCs transplantation than Ang1 and VEGF gene therapy. In conclusion, intramyocardial transplantation of hMSCs improves cardiac function after acute myocardial infarction through enhancement of angiogenesis and myogenesis in the ischemic myocardium. hMSCs are superior to angiogenic growth factor genes for improving myocardial performance in the mouse model of acute myocardial infarction. Transplantation of MSCs may become the future therapy for acute myocardial infarction for myocardial regeneration.

Isolation and characterization of neurogenic mesenchymal stem cells in human scalp tissue.

Recent studies have shown that adult tissues contain stem/ progenitor cells capable of not only generating mature cells of their tissue of origin but also transdifferentiating themselves into other tissue cells. Murine skin-derived precursor cells, for example, have been described as unique, nonmesenchymal-like stem cells capable of mesodermal and ectodermal neurogenic differentiation. Human-derived skin precursors are less well characterized. In this study, the isolation and characterization of adherent, mesenchymal stem cell-like cells from human scalp tissue (hSCPs) are described. hSCPs initially isolated by both medium-selection (ms-hSCPs) and single-cell (c-hSCPs) methods were cultured in medium containing epidermal growth factor and fibroblast growth factor-beta. Cultured ms-hSCPs and c-hSCPs demonstrated a consistent growth rate, continuously replicated in cell culture, and displayed a stable phenotype indistinguishable from each other. Both hSCPs expressed surface antigen profile (CDw90, SH2, SH4, CD105, CD166, CD44, CD49d-e, and HLA class I) similar to that of bone marrow mesenchymal stem cells (BM-MSCs). The growth kinetics, surface epitopes, and differentiation potential of c-hSCP cells were characterized and compared with BM-MSCs. In addition to differentiation along the osteogenic, chondrogenic, and adipogenic lineages, hSCPs can effectively differentiate into neuronal precursors evident by neurogenic gene expression of glial fibrillary acid protein, NCAM, neuron filament-M, and microtubule-associated protein 2 transcripts. Therefore, hSCPs may potentially be a better alternative of BM-MSCs for neural repairing, in addition to their other mesenchymal regenerative capacity. Our study suggests that hSCPs may provide an alternative adult stem cell resource that may be useful for regenerative tissue repair and autotransplantations.