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Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

ABSTRACT: In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

SUBMITTER: El-Kehdy H

PROVIDER: S-EPMC4738699 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

El-Kehdy Hoda H Pourcher Guillaume G Zhang Wenwei W Hamidouche Zahia Z Goulinet-Mainot Sylvie S Sokal Etienne E Charbord Pierre P Najimi Mustapha M Dubart-Kupperschmitt Anne A

Stem cells international 20160113

In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC) decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs). After isolation from 11-12 gestational weeks' human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblasti ...[more]

PMID: 27057173

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Project description:BACKGROUND:Adult mesenchymal stem cells (MSCs) hold great promise for regenerative medicine because of their self-renewal, multipotency, and trophic and immunosuppressive effects. Due to the rareness and high heterogeneity of freshly isolated MSCs, extensive in-vitro passage is required to expand their populations prior to clinical use; however, senescence usually accompanies and can potentially affect MSC characteristics and functionality. Therefore, a thorough characterization of the variations in phenotype and differentiation potential of in-vitro aging MSCs must be sought. METHODS:Human bone marrow-derived MSCs were passaged in vitro and cultivated with either DMEM-based or ?MEM-based expansion media. Cells were prepared for subculture every 10 days up to passage 8 and were analyzed for cell morphology, proliferative capacity, and surface marker expression at the end of each passage. The gene expression profile and adipogenic and osteogenic differentiation capability of MSCs at early (passage 4) and late (passage 8) passages were also evaluated. RESULTS:In-vitro aging MSCs gradually lost the typical fibroblast-like spindle shape, leading to elevated morphological abnormality and inhomogeneity. While the DMEM-based expansion medium better facilitated MSC proliferation in the early passages, the cell population doubling rate reduced over time in both DMEM and ?MEM groups. CD146 expression decreased with increasing passage number only when MSCs were cultured under the DMEM-based condition. Senescence also resulted in MSCs with genetic instability, which was further regulated by the medium recipe. Regardless of the expansion condition, MSCs at both passages 4 and 8 could differentiate into adipocyte-like cells whereas osteogenesis of aged MSCs was significantly compromised. For osteogenic induction, use of the ?MEM-based expansion medium yielded longer osteogenesis and better quality. CONCLUSIONS:Human MSCs subjected to extensive in-vitro passage can undergo morphological, phenotypic, and genetic changes. These properties are also modulated by the medium composition employed to expand the cell populations. In addition, adipogenic potential may be better preserved over osteogenesis in aged MSCs, suggesting that MSCs at early passages must be used for osteogenic differentiation. The current study presents valuable information for future basic science research and clinical applications leading to the development of novel MSC-based therapeutic strategies for different diseases.

Project description:BackgroundDue to their multipotency and ability for self-renewal, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) hold great promise for generating hepatocytes. Previous research has successfully generated hepatocytes from early-passage [i.e., passage (P)3] hUC-MSCs; however, the populations of early-passage cells are limited, and these cells cannot produce sufficient functional hepatocytes for large-scale application in clinical therapy. Thus, a thorough investigation of the hepatic differentiation potential of in vitro-aged hUC-MSCs is needed.MethodshUC-MSCs were passaged in vitro and subcultured every 3 days up to P8, and their morphology, proliferative capacity, liver-specific marker expression, and liver function at the end of each passage were analyzed. The efficiency of the hepatogenic differentiation of hUC-MSCs driven by a functional hit 1 (FH1)-based strategy at different passages was also evaluated.ResultsThe in vitro-aged hUC-MSCs gradually displayed morphological inhomogeneity, had reduced proliferative capability, and exhibited senescent properties while maintaining adipogenic and osteogenic differentiation potential. Additionally, senescence also decreased the expression of messenger RNA (mRNA) levels in albumin (ALB) and alpha 1-antitrpsin (A1AT) in these cells and their relative protein expression, which is the marker of a mature hepatocyte. The liver function of the in vitro-aged hUC-MSCs also deteriorated gradually. Finally, the percentage of hepatocyte-like cells (HLCs) generated from in vitro-aged hUC-MSCs reduced significantly, and the mature hepatocyte functions, such as ALB secretion, glycogen synthesis, low-density lipoprotein (LDL) intake, and indocyanine green (ICG) uptake, also changed.ConclusionshUC-MSCs possess mature hepatocytes' specific markers and functions, which change gradually as they undergo cell senescence. Due to the loss of these properties within in vitro subcultures, the hepatic differentiation efficiency of in vitro-aged hUC-MSCs decreased dramatically in the late passage (P8). The current study provides valuable information can inform future research on liver disease.

Project description:Stem cell therapy for the treatment of tendon injury is an emerging clinical practice in the fields of human and veterinary sports medicine; however, the therapeutic benefit of intralesional transplantation of mesenchymal stem cells in tendonitis cases is not well designed. Questions persist regarding the overall tenogenic potential and efficacy of this treatment alone. In this study, we aimed to isolate a rat mesenchymal stem cell lineage for in vitro and in vivo use, to assess the effects of growth factor exposure in vitro on cell morphology, behavior, and tendon-associated glycoprotein production, and to assess the therapeutic potential of intralesional stem cells, as a function of dose, in vivo. First, rat adipose-derived (rAdMSC) and bone marrow-derived (rBMSC) stem cell lineages were isolated, characterized with flow cytometric analysis, and compared in terms of proliferation (MTS assay) and cellular viability (calcein AM staining). Rat AdMSCs displayed superior proliferation and more homogenous CD 73, CD 44H, and CD 90 expression as compared to rBMSC. Next, the tenogenic differentiation potential of the rAdMSC lineage was tested in vitro through isolated and combined stimulation with reported tenogenic growth factors, transforming growth factor (TGF)-β3 and connective tissue growth factor (CTGF). We found that the most effective tenogenic factor in terms of cellular morphologic change, cell alignment/orientation, sustained cellular viability, and tendon-associated glycoprotein upregulation was TGFβ3, and we confirmed that rAdMSC could be induced toward a tenogenic lineage in vitro. Finally, the therapeutic potential of rAdMSCs as a function of dose was assessed using a rat acute Achilles tendon injury model. Amounts of 5 × 105 (low dose) and 4 × 106 (high dose) were used. Subjectively, on the gross morphology, the rAdMSC-treated tendons exhibited fewer adhesions and less scar tissue than the control tendons; however, regardless of the rAdMSC dose, no significant differences in histological grade or tissue collagen I deposition were noted between the rAdMSC-treated and control tendons. Collectively, rAdMSCs exhibited appropriate stem cell markers and tenogenic potential in vitro, but the clinical efficacy of intralesional implantation of undifferentiated cells in acute tendonitis cases could not be proven. Further investigation into complementary therapeutics or specialized culture conditions prior to implantation are warranted.

Dataset Information

Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

Publications

Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation.

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