Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo

Embryonic stem cells (ES cells), bone marrow-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, and hepatic stem cells in liver have been known as a useful source that can induce to differentiate into hepatocytes. In this study, we examined whether human adipose tissue-derived stromal cells (hADSC) can differentiate into hepatic lineage in vitro. hADSC, that were induced to differentiate into hepatocyte-like cells by the treatment of HGF and OSM, had morphology similar to hepatocytes. Addition of DMSO enhanced differentiation into hepatocytes. RT-PCR and immunocytochemical analysis showed that hADSC express albumin and alpha-fetoprotein during differentiation. Differentiated hADSC showed LDL uptake and production of urea. Additionally, transplanted hADSC to CCl4-injured SCID mouse model were able to be differentiated into hepatocytes and they expressed albumin in vivo. Mesenchymal stem cells isolated from human adipose tissue are immunocompatible and are easily isolated. Therefore, hADSC may become an alternative source to hepatocyte regeneration or liver cell transplantation.     

Characterization and hepatogenic differentiation of mesenchymal stem cells from human amniotic fluid and human bone marrow: a comparative study

Since stem cells can differentiate into hepatocyte, stem cell-based therapy becomes a potential alternative treatment for terminal liver diseases. However, an appropriate source of human mesenchymal stem cells (hMSCs) for hepatocytes has not yet been clearly elucidated. The aim of the present study was to investigate the in vitro biological characterization and hepatic differentiation potential of human amniotic fluid-derived mesenchymal stem cells (AF-hMSCs) and human bone marrow-derived mesenchymal stem cells (BM-hMSCs). Our results show that AF-hMSCs possess higher proliferation and self-renewal capacity than BM-hMSCs. Cytogenetic studies indicate that AF-hMSCs are as genetically stabile as BM-hMSCs. Following incubation with specific hepatogenic agents, AF-hMSCs showed a higher hepatic differentiation potential than BM-hMSCs. Expression of several liver-specific markers was significantly greater in AF-hMSCs than in BM-hMSCs, as shown by real time RT-PCR and immunofluorescence (IF). In conclusion, AF-hMSCs possess superior potential for hepatic differentiation, making them more suitable for diverse terminal liver diseases.     

Transdifferentiation of mesenchymal stem cells derived from human fetal lung to hepatocyte-like cells

The great shortage of human hepatic cells makes it desirable to generate extrahepatic stem or precursor cells. In recent years, it has been reported that human multipotential mesenchymal stem cells (hMSCs) differentiate into hepatocyte-like cells. The fetal lung is one of the largest organs containing many MSCs that can be easily obtained. Whether MSCs from fetal lung can differentiate into hepatocytes or bile duct cells is an important issue in basic medicine and clinical application. We isolated fetal lung cells, and expanded and analyzed them. At passage 4, their morphologic, immunophenotyping and cytokine secretions were similar to adult bone marrow-derived MSCs. We conclude that these cells from fetal lung are MSCs, indicating that human fetal lung is an ideal source of hMSCs. hMSCs from fetal lung induced in special differentiation medium showed homogeneous and small polygonal endothelial-like morphology, expressing weak mRNA, as well as Alb and AFP. This implies that hMSCs from fetal lung can differentiate into hepatocyte-like cells.     

Human amniotic fluid-derived stem cells can differentiate into hepatocyte-like cells in vitro and in vivo

Although human amniotic fluid is an attractive source of multipotent stem cells, the potential of amniotic fluid stem cells (AFSCs) to differentiate into hepatic cells has not been extensively evaluated. In this study, we examined whether human AFSCs can differentiate into a hepatic cell lineage in vitro and in vivo. After being treated with cytokines (fibroblast growth factor 4, basic fibroblast growth factor, hepatocyte growth factor, and oncostatin), AFSCs developed a morphology similar to that of hepatocytes. RT-PCR and immunofluorescence analysis showed that the treated AFSCs expressed the hepatocyte-specific markers albumin, cytokeratin 18, and alpha-fetoprotein. The differentiated cells also developed hepatocyte-specific functions, i.e., they secreted albumin, absorbed indocyanine green, and stored glycogen. When transplanted into CCl(4)-injured immunodeficient mice, undifferentiated AFSCs were integrated into the liver tissue, and they expressed markers characteristic of mature human hepatocytes. Although integration of AFSCs into the liver was limited (0.1-0.3% of hepatocytes), histological analysis showed that the recipient mice recovered more rapidly from CCl(4) injury than CCl(4)-injured mice that did not receive AFSCs. AFSCs can differentiate into hepatocyte-like cells in vitro and in vivo and can represent an easily accessible source of progenitor cells for hepatocyte regeneration and liver cell transplantation.     

Integration of human mesenchymal stem cells into the Wolffian duct in chicken embryos

Developing animal embryos have been providing human mesenchymal stem cells (hMSCs) with an appropriate environment for their differentiation between species. We previously demonstrated that hMSCs transplanted into the metanephric mesenchyme region of rat embryos differentiate into kidney-specific cells. Here, we assessed whether hMSCs are competent to differentiate into precursors of the collecting duct system when they are transplanted into the ureteric bud progenitor region of chicken embryos that are easier to be manipulated and cultured than mammalian embryos. When chicken Pax2-expressing hMSCs were transplanted into the chicken ureteric bud progenitor region, they migrated caudally with the elongating Wolffian duct and then were integrated into the Wolffian duct epithelia. Also, chicken Pax2-expressing hMSCs started to express human LIM1 after their integration into the Wolffian duct epithelia. These results suggest that chicken Pax2-expressing hMSCs can be competent to differentiate into the Wolffian duct cells by the influence of chicken local signals.     

Cardiac potential of stem cells from whole human umbilical cord tissue

We investigated the role of stem cells from human umbilical cord tissue in cardiomyocyte regeneration. The umbilical cord stem cells were initially characterized and differentiated in a myocardial differentiation medium containing 5‐azacytidine for 24 h. Differentiation into cardiomyocytes was determined by expression of cardiac specific markers, like cardiac α‐actin, connexin43, myosin, Troponin T, and ultrastructural analysis. In vivo, the transplanted umbilical cord stem cells were sprouting from local injection and differentiated into cardiomyocyte‐like cells in a rat myocardial infarction model. Echocardiography revealed increasing left ventricular function after umbilical cord stem cell transplantation. These results demonstrate that umbilical cord stem cells can differentiate into cardiomyocyte‐like cells both in vitro and in vivo. Therefore, human umbilical cord might represent a source of stem cells useful for cellular therapy and myocardial tissue engineering. Future studies are required to determine the molecular signaling mechanisms responsible for this phenomenon. J. Cell. Biochem. 107: 926–932, 2009. © 2009 Wiley‐Liss, Inc.     

Establishment of lentiviral-vector-mediated model of human alpha-1 antitrypsin delivery into hepatocyte-like cells differentiated from mesenchymal stem cells

Alpha-1 antitrypsin (AAT) deficiency is a lethal hereditary disorder characterized by a severe diminution in plasma levels of AAT leading to progressive liver dysfunction. Since mesenchymal stem cells can differentiate into hepatocyte-like cells they offer a potential unlimited source in autologous transplant procedures. The transfer of genetically modified hepatocyte cells derived from hMSCs into the body constitutes a novel paradigm of coupling cell therapy with gene therapy for this disease. hMSCs were isolated by density gradient centrifugation and plastic adherence. Hepatic differentiation was induced by exposing hMSC to induction medium for up to 21 days. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR and immunocytochemistry. The chimeric AAT-Jred transgene was transferred to differentiated cells using a lentiviral vector and its expression was visualized by fluorescent microscopy. Flow cytometric analysis confirmed that hMSCs were obtained. Major hepatocyte marker genes expression were confirmed by RT-PCR and immunocytochemistry. AAT gene was successfully introduced into hepatocyte-like cells differentiated from hMSCs. This established system could be suitable for generation of hMSC derived hepatocyte-like cells containing the normal AAT gene, thus offering a potential in vitro source of cells for transplantation therapy of liver diseases in AAT-deficient patients.     

Human mesenchymal stem cells - current trends and future prospective

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton''s jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.     

Human bone marrow mesenchymal stem cells are resistant to HBV infection during differentiation into hepatocytes in vivo and in vitro

Hepatocyte-like cells induced from bone marrow mesenchymal stem cells (BMSCs) recover liver function in animal models with liver failure. Our initial findings revealed that human BMSCs improved liver function in hepatitis B patients with end stage liver disease. However, the susceptibility of BMSCs to HBV infection during induction toward hepatocytes remains unknown. We have assessed whether BMSCs-derived hepatocyte-like cells can function like liver cells and be infected by HBV. A new and efficient way to direct the differentiation of BMSCs into functional hepatocytes was developed. BMSCs obtained from hepatitis B patients were induced to differentiate into hepatocytes through exposure to HGF, FGF-4, and EGF. After 6 days of exposure, BMSCs-derived hepatocyte-like cells that expressed a subset of hepatic genes and showed hepatic functions were obtained. HBV was used to infect the differentiated cells, and subsequently these cells were assayed for the presence of HBeAg, HBsAg, and HBV DNA. BMSCs proved resistant to HBV infection, both in vitro and during differentiation into hepatocytes in vitro. This demonstrates that BMSCs are resistant to HBV infection. BMSCs are viable for transplantation and should facilitate further research exploring the in vivo HBV-resistance of the hepatocytes derived from BMSCs after transplantation, a characteristic that could form the basis for hepatocyte transplantation.     

Transplants of Human Mesenchymal Stem Cells Improve Functional Recovery After Spinal Cord Injury in the Rat

Human mesenchymal stem cells (hMSCs) derived from adult bone marrow represent a potentially useful source of cells for cell replacement therapy after nervous tissue damage. They can be expanded in culture and reintroduced into patients as autografts or allografts with unique immunologic properties. The aim of the present study was to investigate (i) survival, migration, differentiation properties of hMSCs transplanted into non-immunosuppressed rats after spinal cord injury (SCI) and (ii) impact of hMSC transplantation on functional recovery. Seven days after SCI, rats received i.v. injection of hMSCs (2×10⁶ in 0.5 mL DMEM) isolated from adult healthy donors. Functional recovery was assessed by Basso–Beattie–Bresnahan (BBB) score weekly for 28 days. Our results showed gradual improvement of locomotor function in transplanted rats with statistically significant differences at 21 and 28 days. Immunocytochemical analysis using human nuclei (NUMA) and BrdU antibodies confirmed survival and migration of hMSCs into the injury site. Transplanted cells were found to infiltrate mainly into the ventrolateral white matter tracts, spreading also to adjacent segments located rostro-caudaly to the injury epicenter. In double-stained preparations, hMSCs were found to differentiate into oligodendrocytes (APC), but not into cells expressing neuronal markers (NeuN). Accumulation of GAP-43 regrowing axons within damaged white matter tracts after transplantation was observed. Our findings indicate that hMSCs may facilitate recovery from spinal cord injury by remyelinating spared white matter tracts and/or by enhancing axonal growth. In addition, low immunogenicity of hMSCs was confirmed by survival of donor cells without immunosuppressive treatment.     

Growth factor-defined culture medium for human mesenchymal stem cells

Human bone marrow-derived mesenchymal stem cells (hMSCs) are potential cellular sources of therapeutic stem cells as they have the ability to proliferate and differentiate into a wide array of mesenchymal cell types such as osteoblasts, chondroblasts and adipocytes. hMSCs have been used clinically to treat patients with graft vs. host disease, osteogenesis imperfect, or alveolar cleft, suggesting that transplantation of hMSCs is comparatively safe as a stem cell-based therapy. However, conventional culture medium for hMSCs contains fetal bovine serum (FBS). In the present study, we developed a growth factor-defined, serum-free medium for culturing hMSCs. Under these conditions, TGF-beta1 promoted proliferation of hMSCs. The expanded hMSC population expressed the human pluripotency markers SSEA-3, -4, NANOG, OCT3/4 and SOX2. Furthermore, double positive cells for SSEA-3 and a mesenchymal cell marker, CD105, were detected in the population. The potential to differentiate into osteoblasts and adipocytes was confirmed. This work provides a useful tool to understand the basic biological properties of hMSCs in culture.     

A set of microRNAs mediate direct conversion of human umbilical cord lining-derived mesenchymal stem cells into hepatocytes

In a previous study, we elucidated the specific microRNA (miRNA) profile of hepatic differentiation. In this study, we aimed to clarify the instructive role of six overexpressed miRNAs (miR-1246, miR-1290, miR-148a, miR-30a, miR-424 and miR-542-5p) during hepatic differentiation of human umbilical cord lining-derived mesenchymal stem cells (hMSCs) and to test whether overexpression of any of these miRNAs is sufficient to induce differentiation of the hMSCs into hepatocyte-like cells. Before hepatic differentiation, hMSCs were infected with a lentivirus containing a miRNA inhibitor sequence. We found that downregulation of any one of the six hepatic differentiation-specific miRNAs can inhibit HGF-induced hepatic differentiation including albumin expression and LDL uptake. Although overexpression of any one of the six miRNAs alone or liver-enriched miR-122 cannot initiate hepatic differentiation, ectopic overexpression of seven miRNAs (miR-1246, miR-1290, miR-148a, miR-30a, miR-424, miR-542-5p and miR-122) together can stimulate hMSC conversion into functionally mature induced hepatocytes (iHep). Additionally, after transplantation of the iHep cells into mice with CCL4-induced liver injury, we found that iHep not only can improve liver function but it also can restore injured livers. The findings from this study indicate that miRNAs have the capability of directly converting hMSCs to a hepatocyte phenotype in vitro.     

Evidence for a common progenitor of epithelial and mesenchymal components of the liver

Tissues of the adult organism maintain the homeostasis and respond to injury by means of progenitor/stem cell compartments capable to give rise to appropriate progeny. In organs composed by histotypes of different embryological origins (e.g. the liver), the tissue turnover may in theory involve different stem/precursor cells able to respond coordinately to physiological or pathological stimuli. In the liver, a progenitor cell compartment, giving rise to hepatocytes and cholangiocytes, can be activated by chronic injury inhibiting hepatocyte proliferation. The precursor compartment guaranteeing turnover of hepatic stellate cells (HSCs) (perisinusoidal cells implicated with the origin of the liver fibrosis) in adult organ is yet unveiled. We show here that epithelial and mesenchymal liver cells (hepatocytes and HSCs) may arise from a common progenitor. Sca+ murine progenitor cells were found to coexpress markers of epithelial and mesenchymal lineages and to give rise, within few generations, to cells that segregate the lineage-specific markers into two distinct subpopulations. Notably, these progenitor cells, clonally derived, when transplanted in healthy livers, were found to generate epithelial and mesenchymal liver-specific derivatives (i.e. hepatocytes and HSCs) properly integrated in the liver architecture. These evidences suggest the existence of a ‘bona fide'' organ-specific meso-endodermal precursor cell, thus profoundly modifying current models of adult progenitor commitment believed, so far, to be lineage-restricted. Heterotopic transplantations, which confirm the dual differentiation potentiality of those cells, indicates as tissue local cues are necessary to drive a full hepatic differentiation. These data provide first evidences for an adult stem/precursor cell capable to differentiate in both parenchymal and non-parenchymal organ-specific components and candidate the liver as the instructive site for the reservoir compartment of HSC precursors as yet non-localized in the adult.     

Generation of insulin-producing cells from PDX-1 gene-modified human mesenchymal stem cells

Islet cell replacement is considered as the optimal treatment for type I diabetes. However, the availability of human pancreatic islets for transplantation is limited. Here, we show that human bone marrow-derived mesenchymal stem cells (hMSCs) could be induced to differentiate into functional insulin-producing cells by introduction of the pancreatic duodenal homeobox-1 (PDX-1). Recombinant adenoviral vector was used to deliver PDX-1 gene into hMSCs. After being infected with Ad-PDX-1, hMSCs were successfully induced to differentiate into insulin-secreting cells. The differentiated PDX-1+ hMSCs expressed multiple islet-cell genes including neurogenin3 (Ngn3), insulin, GK, Glut2, and glucagon, produced and released insulin/C-peptide in a weak glucose-regulated manner. After the differentiated PDX-1+ hMSCs were transplanted into STZ-induced diabetic mice, euglycemia can be obtained within 2 weeks and maintained for at least 42 days. These findings validate the hMSCs model system as a potential basis for enrichment of human beta cells or their precursors, and a possible source for cell replacement therapy in diabetes.     

Hepatic differentiation from human mesenchymal stem cells on a novel nanofiber scaffold

The emerging fields of tissue engineering and biomaterials have begun to provide potential treatment options for liver failure. The goal of the present study is to investigate the ability of a poly L-lactic acid (PLLA) nanofiber scaffold to support and enhance hepatic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). A scaffold composed of poly L-lactic acid and collagen was fabricated by the electrospinning technique. After characterizing isolated hMSCs, they were seeded onto PLLA nanofiber scaffolds and induced to differentiate into a hepatocyte lineage. The mRNA levels and protein expression of several important hepatic genes were determined using RT-PCR, immunocytochemistry and ELISA. Flow cytometry revealed that the isolated bone marrow-derived stem cells were positive for hMSC-specific markers CD73, CD44, CD105 and CD166 and negative for hematopoietic markers CD34 and CD45. The differentiation of these stem cells into adipocytes and osteoblasts demonstrated their multipotency. Scanning electron microscopy showed adherence of cells in the nanofiber scaffold during differentiation towards hepatocytes. Our results showed that expression levels of liver-specific markers such as albumin, α-fetoprotein, and cytokeratins 8 and 18 were higher in differentiated cells on the nanofibers than when cultured on plates. Importantly, liver functioning serum proteins, albumin and α-1 antitrypsin were secreted into the culture medium at higher levels by the differentiated cells on the nanofibers than on the plates, demonstrating that our nanofibrous scaffolds promoted and enhanced hepatic differentiation under our culture conditions. Our results show that the engineered PLLA nanofibrous scaffold is a conducive matrix for the differentiation of MSCs into functional hepatocyte-like cells. This represents the first step for the use of this nanofibrous scaffold for culture and differentiation of stem cells that may be employed for tissue engineering and cell-based therapy applications.     

Stem/progenitor cells in liver injury repair and regeneration

Morbidity and mortality from cirrhosis is increasing rapidly in the world. Currently, orthotopic liver transplantation is the only definitive therapeutic option. However, its clinical use is limited, because of poor long‐term graft survival, donor organ shortage and high costs associated with the procedure. Stem cell replacement strategies are therefore being investigated as an attractive alternative approach to liver repair and regeneration. In this review we discuss recent preclinical and clinical investigations that explore the therapeutic potential of stem cells in repair of liver injuries. Several types of stem cells. including embryonic stem cells, haematopoietic stem cells and mesenchymal stem cells, can be induced to differentiate into hepatocyte‐like cells by defined culture conditions in vitro. Stem cell transplantation has been shown to significantly improve liver function and increase animal survival in experimentally‐induced liver‐injury models. Moreover, several pilot clinical studies have reported encouraging therapeutic effects in patients treated with stem cells. Although there remain many unresolved issues, the available data support the notion that stem cell technology may lead to the development of effective clinical modalities for human liver diseases.     

Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells in an alginate scaffold

Objectives: Alginate scaffolds are the most frequently investigated biomaterials in tissue engineering. Tissue engineering techniques that generate liver tissue have become important for treatment of a number of liver diseases and recent studies indicate that bone marrow‐derived stem cells (BMSCs) can differentiate into hepatocyte‐like cells. The goal of the study described here, was to examine in vitro hepatic differentiation potential of BMSCs cultured in an alginate scaffold. Materials and methods: To investigate the potential of BMSCs to differentiate into hepatocyte‐like cells, we cultured BMSCs in alginate scaffolds in the presence of specific growth factors including hepatocyte growth factor, epidermal growth factor and fibroblast growth factor‐4. Results: We can demonstrate that alginate scaffolds are compatible for growth of BMSCs and when cultured in alginate scaffolds for several days they display several liver‐specific markers and functions. Specifically, they expressed genes encoding alpha‐foetoprotein, albumin (ALB), connexin 32 and CYP7A1. In addition, these BMSCs produced both ALB and urea, expressed cytokeratin‐18 (CK‐18) and were capable of glycogen storage. Percentage of CK‐18 positive cells, a marker of hepatocytes, was 56.7%. Conclusions: Our three‐dimensional alginate scaffolds were highly biocompatible with BMSCs. Furthermore, culturing induced their differentiation into hepatocyte‐like cells. Therefore, BMSCs cultured in alginate scaffolds may be applicable for hepatic tissue engineering.