Human bone marrow-derived mesenchymal stem cells differentiate into epidermal-like cells in vitro

Human bone marrow-derived mesenchymal stem cells (hMSCs) are a population of pluripotent cells. They can differentiate into different embryonic layer cells as osteoblasts, adipocytes, chondrocytes, myoblasts, neurocytes, etc. However, there are only few reports with regard to differentiate hMSCs into epidermal cells in vitro. In this study, we want to investigate the feasibility of inducing hMSCs into epidermal-like cells under specific medium in vitro. hMSCs in specific inducing medium expressed the early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), the late differentiated marker, the pan-cytokeratin, and another early marker, the beta1-integrin, which up-regulated remarkably in inducing medium. Their morphologies were changed from spindle-like fibroblastic appearances to oblate or irregular shapes under phase contrast microscopy. The hemidesmosome structure was found using the transmission electron microscope. All these data suggested that, under certain conditions, hMSCs have the potential to differentiate into epidermal-like cells. It will be of great accordance in the study of the multipotential property of hMSCs.     

Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro

It was recently reported that pluripotent mesenchymal stem cells (MSCs) in rodent bone marrow (BM) have the capacity to generate insulin-producing cells (IPCs) in vitro. However, little is known about this capacity in human BM-MSCs. We developed a nongenetic method to induce human BM-MSCs to transdifferentiate into IPCs both phenotypically and functionally. BM-MSCs from 12 human donors were sequentially cultured in specially defined conditions. Their differentiation extent toward β-cell phenotype was evaluated systemically. Specifically, after induction human BM-MSCs formed spheroid islet-like clusters containing IPCs, which was further confirmed by dithizone (DTZ) staining and electron microscopy. These IPCs expressed multiple genes related to the development or function of pancreatic β cells (including NKX6.1, ISL-1, Beta2/Neurod, Glut2, Pax6, nestin, PDX-1, ngn3, insulin and glucagon). The coexpression of insulin and c-peptide was observed in IPCs by immunofluorescence. Moreover, they were able to release insulin in a glucose-dependent manner and ameliorate the diabetic conditions of streptozotocin (STZ)-treated nude mice. These results indicate that human BM-MSCs might be an available candidate to overcome limitations of islet transplantation.     

Human umbilical cord-derived mesenchymal stem cells differentiate into epidermal-like cells using a novel co-culture technique

Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) isolated from human umbilical Wharton’s Jelly are a population of primitive and pluripotent cells. In specific conditions, hUCMSCs can differentiate into various cells, including adipocytes, osteoblasts, chondrocytes, neurocytes, and endothelial cells. However, few studies have assessed their differentiation into epidermal cells in vitro. To assess the potential of hUCMSCs to differentiate into epidermal cells, a microporous membrane-based indirect co-culture system was developed in this study. Epidermal stem cells (ESCs) were seeded on the bottom of the microporous membrane, and hUCMSCs were seeded on the top of the microporous membrane. Cell morphology was assessed by phase contrast microscopy, and the expression of early markers of epidermal cell lineage, P63, cytokeratin19 (CK19), and β1-integrin, was determined by immunofluorescence, Western blot, and quantitative real-time PCR (Q-PCR) analyses. hUCMSC morphology changed from spindle-like to oblate or irregular with indirect co-culture with ESCs; they also expressed greater levels P63, CK19, and β1-integrin mRNA and protein compared to the controls (p < 0.01). As compared to normal co-cultures, indirect co-culture expressed significantly greater CK19 protein (p < 0.01). Thus, hUCMSCs may have the capability to differentiate into the epidermal lineage in vitro, which may be accomplished through this indirect co-culture model.     

Adipose tissue-derived mesenchymal stromal cells efficiently differentiate into insulin-producing cells in pancreatic islet microenvironment both in vitro and in vivo

Background aimsDifferentiation or reprogramming of stem cells could be achieved by remodulating the microenvironment, which regulates the fate of cells by soluble factors and contacts. By providing an in vivo-like microenvironment, directional and functional differentiation of stem cells could be achieved in vitro. In this study, the differentiation of mesenchymal stromal cells (MSCs) derived from rat tissues (adipose, rAT; bone marrow, rBM) were analyzed by in vitro and in vivo co-culture experiments. The insulin-producing capacities of islets transplanted under the renal kidney capsule with rAT- and rBM-MSCs were compared and the reduction of hyperglycemia symptoms in rat models was examined.MethodsMSCs prelabeled with green fluorescence protein were co-cultured with islets directly. The insulin production of cells was determined by immunostaining and ELISA. Streptozotocin-induced diabetic rat models were created and MSCs were co-transplanted with the islets under the kidney capsule to confirm the in vitro results.ResultsMSCs were differentiated into insulin-producing cells after 38 days of co-culture, confirmed by insulin and C-peptide stainings. In vivo functional studies revealed that the co-culture of islets with MSCs provided higher differentiation efficiency. The weight gain measurement and glucose tolerance test in the rat group co-transplanted of rAT-MSCs and islets indicate a better recovery than islet-alone transplants and co-transplants of islets and rBM-MSCs.ConclusionsrAT-MSCs could be considered as the cell of choice for cell-based treatment of type 1 diabetes. Because the co-transplantation of islets with MSCs increases the number of insulin-producing cells, this method was suggested for clinical applications.     

Human mesenchymal adipose stromal cells from mature adipocyte fraction

More effective techniques should be employed for isolation of human mesenchymal stromal cells derived from adipose tissue (ADSC), seeking to make adipose tissue biopsies smaller in volume and thus less invasive. In this study, we compared properties of ADSC isolated by several different methods from the same samples of adipose tissue in order to enhance yields of potential ADSC. The mature adipocyte fraction was investigated using the ceiling culture method, including both ceiling and bottom cell fractions, and the control culture method with standard amount of medium. The results were also compared using the stromal vascular fraction from the same samples. The most efficient was the bottom cell population isolated from the mature adipocyte fraction by ceiling culture method. These cells readily differentiated into osteogenic, adipogenic and chondrogenic lineages and, similar to stromal vascular fraction cells, displayed high proliferation potential. Cultures of mature adipocyte fractions with standard amount of medium were considerably less effective. Mature adipocyte fractions yields large quantities of adipose-derived stem cells that have properties comparable with stromal vascular fraction cells suitable for tissue regeneration, especially when only small biopsies can be taken.     

Adult rat bone marrow stromal cells differentiate into Schwann cell-like cells in vitro

Bone marrow stromal cells (MSCs) have the capability of differentiating into mesenchymal and non-mesenchymal lineages. In this study, MSCs isolated from adult Sprague-Dawley rats were cultured to proliferation, followed by in vitro induction under specific conditions. The results demonstrated that MSCs were transdifferentiated into cells with the Schwann cell (SC) phenotypes according to their morphology and immunoreactivities to SC surface markers including S-100, glial fibrillary acidic protein (GFAP) and low-affinity nerve growth factor receptor (p75). Consequently, rat adult MSCs can be induced in vitro to differentiate into SC-like cells, thus developing an abundant and accessible SC reservoir to meet the requirements of constructing tissue engineered nerve grafts for peripheral nerve repair.     

The generation of three-dimensional tissue structures with mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent stem cells, found in the bone-marrow and other adult tissues, which give rise to various cell lineages. Although MSCs are biologically important, and may have widespread therapeutic potential, they are not well-characterised, particularly in terms of their cell surface receptors and in vivo phenotype. We aimed to develop a three-dimensional (3-D) MSC in vitro model, in order to understand the factors involved in the regulation of lineage specification routes. A suitable model, which replicates the MSC microenvironment as accurately as possible, will allow more detailed investigations into the phenotype of the cells. Our MSC spheroids appear to have an enhanced mesenchymal differentiation compared to two-dimensional MSC monolayers. With this in vitro system, it is possible to perform real-time analysis of cellular differentiation status. MSC spheroids may also be amenable for use in high-throughput assays. A more-recent research project aims to generate knockout micro-tissues, based on human 3-D MSCs, as an alternative to animal studies.     

Mesodermal progenitor cells (MPCs) differentiate into mesenchymal stromal cells (MSCs) by activation of Wnt5/calmodulin signalling pathway

Background

Mesenchymal Stromal Cells (MSCs) remain poorly characterized because of the absence of manifest physical, phenotypic, and functional properties in cultured cell populations. Despite considerable research on MSCs and their clinical application, the biology of these cells is not fully clarified and data on signalling activation during mesenchymal differentiation and proliferation are controversial. The role of Wnt pathways is still debated, partly due to culture heterogeneity and methodological inconsistencies. Recently, we described a new bone marrow cell population isolated from MSC cultures that we named Mesodermal Progenitor Cells (MPCs) for their mesenchymal and endothelial differentiation potential. An optimized culture method allowed the isolation from human adult bone marrow of a highly pure population of MPCs (more than 97%), that showed the distinctive SSEA-4⁺CD105⁺CD90^neg phenotype and not expressing MSCA-1 antigen. Under these selective culture conditions the percentage of MSCs (SSEA-4^negCD105⁺CD90^bright and MSCA-1⁺), in the primary cultures, resulted lower than 2%.

Methodology/Principal Finding

We demonstrate that MPCs differentiate to MSCs through an SSEA-4⁺CD105⁺CD90^bright early intermediate precursor. Differentiation paralleled the activation of Wnt5/Calmodulin signalling by autocrine/paracrine intense secretion of Wnt5a and Wnt5b (p<0.05 vs uncondictioned media), which was later silenced in late MSCs (SSEA-4^neg). We found the inhibition of this pathway by calmidazolium chloride specifically blocked mesenchymal induction (ID₅₀ = 0.5 µM, p<0.01), while endothelial differentiation was unaffected.

Conclusion

The present study describes two different putative progenitors (early and late MSCs) that, together with already described MPCs, could be co-isolated and expanded in different percentages depending on the culture conditions. These results suggest that some modifications to the widely accepted MSC nomenclature are required.     

Differentiation of multipotent mesenchymal stromal cells of human bone marrow into cells of cartilage tissue by culturing in three-dimensional OPLA scaffolds

Bone marrow multipotent mesenchymal stromal cells show considerable promise for the engineering of human three-dimensional transplants of cartilage tissue. We demonstrated the directed differentiation of BM MMSC in cells of cartilage tissue by culturing them in OPLA polymer three-dimensional scaffolds in a medium with chondrogenesis inducers. Cells were loaded into porous scaffolds by saturating polymer blocks with a cellular suspension. This was followed by high-speed centrifugation of the matrix-embedded cells and cultivation of the engineering constructs in a condrogenic medium for 28 days. Histological analysis of the three-dimensional transplants derived in vitro showed a uniform distribution of cells in the matrix. Morphologically, the cells were similar to the chondrocyte-like cells of articular cartilage. Immunohistochemical analysis revealed aggrecan and collagen type 2, which are the major markers of chondrogenesis, in the constructs. Preclinical research on immunodeficient mice showed that the engineering transplants were not toxic.     

Human chorionic villus mesenchymal stromal cells reveal strong endothelial conversion properties

Chorion, amnion and villi are reservoirs of mesenchymal stromal cells (StC) and the hypothesis that StC from fetal tissues retain higher plasticity compared to adult StC has been suggested. Aimed at investigating this aspect, a series of in vitro experiments were performed with StC isolated from first trimester human chorionic villi (CVStC). CVStC were cultured in: (i) standard mesenchymal medium (MM) and (ii) AmniomaxII? (AM), specifically designed to grow amnion-derived cells in prenatal diagnostic procedures. Cells were then exposed to distinct differentiation treatments and distinguished according to morphology, immunophenotype and molecular markers. Human StC obtained from adult bone marrow (BMStC) were used as control. CVStC cultured either in MM or AM presented stromal morphology and immunophenotype, were negative for pluripotency factors (Nanog, Oct-4 and Sox-2), lacked detectable telomerase activity and retained high genomic stability. In AM, however, CVStC exhibited a faster proliferation rate compared to BMStC or CVStC kept in MM. During differentiation, CVStC were less efficient than BMStC in acquiring adipocytes and osteocytes features; the cardiomyogenic conversion occurred at low efficiency in both cell types. Remarkably, in the presence of pro-angiogenic factors, CVStC reprogrammed toward an endothelial-like phenotype at significantly higher efficiency than BMStC. This effect was particularly evident in CVStC expanded in AM. Mechanistically, the reduced CVStC expression of anti-angiogenic microRNA could support this process. The present study demonstrates that, despite of fetal origin, CVStC exhibit restricted plasticity, distinct from that of BMStC and predominantly directed toward the endothelial lineage.     

Enhanced differentiation potential of human amniotic mesenchymal stromal cells by using three-dimensional culturing

The therapeutic potential of human amniotic mesenchymal stromal cells (hAMSCs) remains limited because of their differentiation towards mesenchymal stem cells (MSCs) following adherence. The aim of this study was to develop a three-dimensional (3-D) culture system that would permit hAMSCs to differentiate into cardiomyocyte-like cells. hAMSCs were isolated from human amnions of full-term births collected after Cesarean section. Immunocytochemistry, immunofluorescence and flow cytometry analyses were undertaken to examine hAMSC marker expression for differentiation status after adherence. Membrane currents were determined by patch clamp analysis of hAMSCs grown with or without cardiac lysates. Freshly isolated hAMSCs were positive for human embryonic stem-cell-related markers but their marker profile significantly shifted towards that of MSCs following adherence. hAMSCs cultured in the 3-D culture system in the presence of cardiac lysate expressed cardiomyocyte-specific markers, in contrast to those maintained in standard adherent cultures or those in 3-D cultures without cardiac lysate. hAMSCs cultured in 3-D with cardiac lysate displayed a cardiomyocyte-like phenotype as observed by membrane currents, including a calcium-activated potassium current, a delayed rectifier potassium current and a Ca²⁺-resistant transient outward K⁺ current. Thus, although adherence limits the potential of hAMSCs to differentiate into cardiomyocyte-like cells, the 3-D culture of hAMSCs represents a more effective method of their culture for use in regenerative medicine.