Transformation of human umbilical mesenchymal cells into neurons in vitro

Neuronal transplantation has provided a promising approach for treating neurodegenerative diseases. Recently, efforts have been directed at in vitro induction of various stem cells to transform into neurons. We report the first successful quantities in an in vitro attempt at directing the transformation into neurons of human umbilical mesenchymal cells, which are capable of rapid proliferation in vitro and are easily available. When cultured in neuronal conditioned medium, human umbilical mesenchymal cells started to express neuron-specific proteins such as NeuN and neurofilament (NF) on the 3rd day and exhibited retraction of the cell body, elaboration of processes, clustering of cells and expression of functional mRNA responsible for the synthesis of subunits of the kainate receptor and glutamate decarboxylase on the 6th day. Between the 9th and 12th days, the percentage of human umbilical mesenchymal cells expressing NF was as high as 87%, while functionality was demonstrated by glutamate invoking an inward current. At this stage, cells were differentiated into mature neurons in the postmitosis phase.     

Transformation of fibroblasts into endothelial cells during angiogenesis

Light-and electron-microscopic autoradiography have been used to study fibroblast transformation into endothelial cells in the formation of new blood vessels during wound healing in rabbit ear chambers. When cultured fibroblasts labeled with tritium thymidine were transplanted autologously into the chambers, newly formed blood vessels contained endothelial cells labeled with tritium thymidine. This result suggests that fibroblasts play a pivotal role in angiogenesis, as progenitors of endothelial cells in newly formed blood vessels.     

Transformation/neodifferentiation of human skin fibroblasts by acute oncornaviruses and dexamethasone

We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells.     

Oxidative stress response of human fibroblasts and endometrial mesenchymal stem cells

Human mesenchymal stem cells are a promising cell source for tissue engineering. During transplantation, they may be subjected to oxidative stress due to unfavorable cellular microenvironment characterized by an increased level of reactive oxygen species. Recently, we have demonstrated that oxidative stress response of human mesenchymal stem cells derived from endometrium (hMESCs) depends on the oxidizer concentration. The duration of cell treatment with an oxidizer also may play an important role. In this study, we investigated the dependence of the cell response on H₂O₂ treatment duration. The effects of high H₂O₂ doses on hMESCs and human lung embryonic fibroblasts were compared. In both cell types, H₂O₂ treatment for 60 min caused multiphase cell cycle arrest, with dose-dependent cell death occurring equally in all phases of the cell cycle. However, the cell death dynamics in hMESCs and fibroblasts were different. Interestingly, in both cell types, shortening of H₂O₂ treatment from 60 to 10 min induced growth retardation, G1-phase cell accumulation, and cell size increase. Collectively, these findings suggest that there is induction of premature senescence. Thus, shortening of oxidative stress induced in human endometrial stem cells and embryonic fibroblasts by high H₂O₂ doses enables one to modulate cellular response as both cell death and premature senescence.     

N-Cadherin is expressed on human hematopoietic progenitor cells and mediates interaction with human mesenchymal stromal cells

Specific cell–cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human–human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and β-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.     

Adult human fibroblasts are potent immunoregulatory cells and functionally equivalent to mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSC) have potent immunosuppressive properties and have been advocated for therapeutic use in humans. The nature of their suppressive capacity is poorly understood but is said to be a primitive stem cell function. Demonstration that adult stromal cells such as fibroblasts (Fb) can modulate T cells would have important implications for immunoregulation and cellular therapy. In this report, we show that dermal Fb inhibit allogeneic T cell activation by autologously derived cutaneous APCs and other stimulators. Fb mediate suppression through soluble factors, but this is critically dependent on IFN-gamma from activated T cells. IFN-gamma induces IDO in Fb, and accelerated tryptophan metabolism is at least partly responsible for suppression of T cell proliferation. T cell suppression is reversible, and transient exposure to Fb during activation reprograms T cells, increasing IL-4 and IL-10 secretion upon restimulation. Increased Th2 polarization by stromal cells is associated with amelioration of pathological changes in a human model of graft-vs-host disease. Dermal Fb are highly clonogenic in vitro, suggesting that Fb-mediated immunosuppression is not due to outgrowth of rare MSC, although dermal Fb remain difficult to distinguish from MSC by phenotype or transdifferentiation capacity. These results suggest that immunosuppression is a general property of stromal cells and that dermal Fb may provide an alternative and accessible source of cellular therapy.     

Gene and microRNA expression signatures of human mesenchymal stromal cells in comparison to fibroblasts

Human mesenchymal stromal cells (MSCs) offer great hope for the treatment of tissue degenerative and immune diseases, but their phenotypic similarity to dermal fibroblasts may hinder robust cell identification and isolation from diverse tissue harvests. To identify genetic elements that can reliably discriminate MSCs from fibroblasts, we performed comparative gene and microRNA expression profiling analyses with genome-wide oligonucleotide microarrays. When taken globally, both gene and microRNA expression profiles of MSCs were highly similar to those of fibroblasts, accounting well for their extensive phenotypic and functional overlaps. Scattered expression differences were pooled to yield an MSC-specific molecular signature, consisting of 64 genes and 21 microRNAs whose expressions were at least 10-fold and two-fold higher, respectively, in MSCs compared with fibroblasts. Genes either encoding transmembrane proteins or associated with tumors were relatively abundant in this signature. These data should provide the molecular basis not only for the discovery of novel diagnostic markers discriminating MSCs from fibroblasts, but also for further studies on MSC-specific signaling mechanisms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sohyun Bae and Jung Hoon Ahn contributed equally to this work. This research was supported in part by a grant (SC-2140) from Stem Cell Research Center of the 21st Century Frontier Research Program, and in part by Korea Science and Engineering Foundation grant (M10641000037) funded by the Ministry of Education, Science and Technology, Republic of Korea.     

Cultured human skin fibroblasts and arterial cells produce a labile platelet-inhibitory prostaglandin

Human skin fibroblasts and cells cultured from human arterial smooth muscle produce a platelet-inhibitory prostaglandin in response to mechanical trauma. This prostaglandin is synthesized from an endogenous precursor rather than exogenous cyclic endoperoxides; it differs from PGE₁ and PGD₂ and resembles PGI₂ (prostacyclin) in its stability properties, being stable at pH ≥ 8.5 and labile at pH 7.4 and below. The prostaglandin synthesis pathway in these cultured cells is less sensitive to inhibition by aspirin than that in human platelets.     

Therapeutic effect of human mesenchymal stem cells in skin after radiation damage

Over 50% of all cancer patients presently receive radiotherapy at one stage in their treatment course. Inevitably skin is one of the most frequently damaged tissue due to its localization and constant turn-over. Our present goal is to reduce radiation-induced complications in human skin through stem cell therapy, particulary in human epidermis. Mesenchymal Stem Cells (MSCs) have been shown to be multipotent cells able to engraft in many tissues after injury. Herein, we isolated human MSCs and tested their capability to improve skin wound healing after irradiation. This potential was assessed in NOD/SCID mice which received 30 Gy locally on the thigh. This dose caused within 3 weeks local epidermis necrosis which was repaired within 13 weeks. MSCs were intravenously injected in irradiated mice 24 hours after exposure. Clinical scoring throughout 6 weeks gave indications that human MSCs reduced the extent of damage and accelerated the wound healing process. We show by quantitative qPCR and histological studies the presence of human MSCs derived cells into the scar. Human MSCs homed to the damaged skin and participated to the wound healing process. These results open prospects for cellular therapy by MSCs in irradiated epithelial tissues and could be extended to the whole general field of cutaneous cicatrization, particularly after burns.     

Differentiation of human fetal mesenchymal stem cells into cells with an oligodendrocyte phenotype

The potential of mesenchymal stem cells (MSC) to differentiate into neural lineages has raised the possibility of autologous cell transplantation as a therapy for neurodegenerative diseases. We have identified a population of circulating human fetal mesenchymal stem cells (hfMSC) that are highly proliferative and can readily differentiate into mesodermal lineages such as bone, cartilage, fat and muscle. Here, we demonstrate for the first time that primary hfMSC can differentiate into cells with an oligodendrocyte phenotype both in vitro and in vivo. By exposing hfMSC to neuronal conditioned medium or by introducing the pro-oligodendrocyte gene, Olig-2, hfMSC adopted an oligodendrocyte-like morphology, expressed oligodendrocyte markers and appeared to mature appropriately in culture. Importantly we also demonstrate the differentiation of a clonal population of hfMSC into both mesodermal (bone) and ectodermal (oligodendrocyte) lineages. In the developing murine brain transplanted hfMSC integrated into the parenchyma but oligodendrocyte differentiation of these naïve hfMSC was very low. However, the proportion of cells expressing oligodendrocyte markers increased significantly (from 0.2% to 4%) by pre-exposing the cells to differentiation medium in vitro prior to transplantation. Importantly, the process of in vivo differentiation occurred without cell fusion. These findings suggest that hfMSC may provide a potential source of oligodendrocytes for study and potential therapy.     

The differentiation of human placenta-derived mesenchymal stem cells into dopaminergic cells in vitro

Mesenchymal stem cells (MSCs) constitute an interesting cellular source to promote brain regeneration after Parkinson’s disease. MSCs have significant advantages over other stem cell types, and greater potential for immediate clinical application. The aim of this study was to investigate whether MSCs from the human placenta could be induced to differentiate into dopaminergic cells. MSCs from the human placenta were isolated by digestion and density gradient fractionation, and their cell surface glycoproteins were analyzed by flow cytometry. These MSCs were cultured under conditions promoting differetiation into adipocytes and osteoblasts. Using a cocktail that includes basic fibroblast growth factor (bFGF), all trans retinoic acid (RA), ascorbic acid (AA) and 3-isobutyl-1-methylxanthine (IBMX), the MSCs were induced in vitro to become dopamine (DA) neurons. Then, the expression of the mRNA for the Nestin and tyrosine hydroxylase (TH) genes was assayed via RT-PCR. The expression of the Nestin, dopamine transporter (DAT), neuronal nuclear protein (NeuN) and TH proteins was determined via immunofluorescence. The synthesized and secreted DA was determined via ELISA. We found that MSCs from the human placenta exhibited a fibroblastoid morphology. Flow cytometric analyses showed that the MSCs were positive for CD44 and CD29, and negative for CD34, CD45, CD106 and HLA-DR. Moreover, they could be induced into adipocytes and osteocytes. When the MSCs were induced with bFGF, RA, AA and IBMX, they showed a change in morphology to that of neuronal-like cells. The induced cells expressed Nestin and TH mRNA, and the Nestin, DAT, NeuN and TH proteins, and synthesized and secreted DA. Our results suggest that MSCs from the human placenta have the ability to differentiate into dopaminergic cells.     

Hyaluronidase expression in human skin fibroblasts

Hyaluronidase activity has been detected for the first time in normal human dermal fibroblasts (HS27), as well as in fetal fibroblasts (FF24) and fibrosarcoma cells (HT1080). Enzymatic activity was secreted predominantly into the culture media, with minor amounts of activity associated with the cell layer. In both classes of fibroblasts, hyaluronidase expression was confluence-dependent, with highest levels of activity occurring in quiescent, post-confluent cells. However, in the fibrosarcoma cell cultures, expression was independent of cell density. The enzyme had a pH optimum of 3.7 and on hyaluronan substrate gel zymography, activity occurred as a single band corresponding to an approximate molecular size of 57 kDa. The enzyme could be immunoprecipitated in its entirety using monoclonal antibodies raised against Hyal-1, human plasma hyaluronidase. PCR confirmed that fibroblast hyaluronidase was identical to Hyal-1. The conclusion by previous investigators using earlier technologies that fibroblasts do not contain hyaluronidase activity should be reevaluated.