Generation of Human Epidermis-Derived Mesenchymal Stem Cell-like Pluripotent Cells and their reprogramming in mouse chimeras

Stem cells can be derived from the embryo (embryonic stem cells, ESCs), from adult tissues (adult stem cells, ASCs), and by induction of fibroblasts (induced pluripotent stem cells, iPSs). Ethical problems, immunological rejection, and difficulties in obtaining human tissues limit the use of ESCs in clinical medicine. Induced pluripotent stem cells are difficult to maintain in vitro and carry a greater risk of tumor formation. Furthermore, the complexity of maintenance and propagation is especially difficult in the clinic. Adult stem cells can be isolated from several adult tissues and present the possibility of self-transplantation for the clinical treatment of a variety of human diseases. Recently, several ASCs have been successfully isolated and cultured in vitro, including hematopoietic stem cells (HSCs) , mesenchymal stem cells (MSCs), epidermis stem cells, neural stem cells (NSCs), adipose-derived stem cells (ADSCs), islet stem cells, and germ line stem cells. Human mesenchymal stem cells originate mainly from bone marrow, cord blood, and placenta, but epidermis-derived MSCs have not yet been isolated. We isolated small spindle-shaped cells with strong proliferative potential during the culture of human epidermis cells and designed a medium to isolate and propagate these cells. They resembled MSCs morphologically and demonstrated pluripotency in vivo; thus, we defined these cells as human epidermis-derived mesenchymal stem cell-like pluripotent cells (hEMSCPCs). These hEMSCPCs present a possible new cell resource for tissue engineering and regenerative medicine

Milieu-adopted in vitro and in vivo differentiation of mesenchymal tissues derived from different adult human CD34-negative progenitor cell clones

Adult mesenchymal stem cells with multilineage differentiation potentially exist in the bone marrow, but have also been isolated from the peripheral blood. The differentiation of stem cells after leaving their niches depends predominately on the local milieu and its new microenvironment, and is facilitated by soluble factors but also by the close cell-cell interaction in a three-dimensional tissue or organ system. We have isolated CD34-negative, mesenchymal stem cell lines from human bone marrow and peripheral blood and generated monoclonal cell populations after immortalization with the SV40 large T-antigen. The cultivation of those adult stem cell clones in an especially designed in vitro environment, including self-constructed glass capillaries with defined growth conditions, leads to the spontaneous establishment of pleomorphic three-dimensional cell aggregates ( spheroids) from the monoclonal cell population, which consist of cells with an osteoblast phenotype and areas of mineralization along with well-vascularized tissue areas. Modifications of the culture conditions favored areas of bone-like calcifications. After the transplantation of the at least partly mineralized human spheroids into different murine soft tissue sites but also a dorsal skinfold chamber, no further bone formation could be observed, but angiogenesis and neovessel formation prevailed instead, enabling the transplanted cells and cell aggregates to survive. This study provides evidence that even monoclonal adult human CD34-negative stem cells from the bone marrow as well as peripheral blood can potentially differentiate into different mesenchymal tissues depending on the local milieu and responding to the needs within the microenvironment. Copyright (C) 2005 S. Karger AG, Basel

Perivascular mesenchymal stem cells in the adult human brain: a future target for neuroregeneration?

Perivascular adult stem cells have been isolated from several tissues, including the adult human brain. They have unique signatures resembling both pericytes and mesenchymal stem cells. Understanding the nature of these cells in their specific vascular niches is important to determine their clinical potential as a new adult stem cell source. Indeed, they have promising features in vitro in terms of multipotency, immunomodulation and secretion of growth factors and cytokines. However, their in vivo function is less known as yet. Recent emerging data show a crucial role of perivascular mesenchymal stem cells in tissue homeostasis and repair. Furthermore, these cells may play an important role in adult stem cell niche regulation and in neurodegeneration. Here we review the recent literature on perivascular mesenchymal stem cells, discuss their different in vitro functions and highlight especially the specific properties of brain-derived perivascular mesenchymal stem cells. We summarize current evidence that suggests an important in vivo function of these cells in terms of their regenerative potential that may indicate a new target cell for endogenous tissue regeneration and repair

The Adult Human Brain Harbors Multipotent Perivascular Mesenchymal Stem Cells

Blood vessels and adjacent cells form perivascular stem cell niches in adult tissues. In this perivascular niche, a stem cell with mesenchymal characteristics was recently identified in some adult somatic tissues. These cells are pericytes that line the microvasculature, express mesenchymal markers and differentiate into mesodermal lineages but might even have the capacity to generate tissue-specific cell types. Here, we isolated, purified and characterized a previously unrecognized progenitor population from two different regions in the adult human brain, the ventricular wall and the neocortex. We show that these cells co-express markers for mesenchymal stem cells and pericytes in vivo and in vitro, but do not express glial, neuronal progenitor, hematopoietic, endothelial or microglial markers in their native state. Furthermore, we demonstrate at a clonal level that these progenitors have true multilineage potential towards both, the mesodermal and neuroectodermal phenotype. They can be epigenetically induced in vitro into adipocytes, chondroblasts and osteoblasts but also into glial cells and immature neurons. This progenitor population exhibits long-term proliferation, karyotype stability and retention of phenotype and multipotency following extensive propagation. Thus, we provide evidence that the vascular niche in the adult human brain harbors a novel progenitor with multilineage capacity that appears to represent mesenchymal stem cells and is different from any previously described human neural stem cell. Future studies will elucidate whether these cells may play a role for disease or may represent a reservoir that can be exploited in efforts to repair the diseased human brain

Epithelial-Mesenchymal Transition in Cells Expanded In Vitro from Lineage-Traced Adult Human Pancreatic Beta Cells

BACKGROUND: In-vitro expansion of functional beta cells from adult human islets is an attractive approach for generating an abundant source of cells for beta-cell replacement therapy of diabetes. Using genetic cell-lineage tracing we have recently shown that beta cells cultured from adult human islets undergo rapid dedifferentiation and proliferate for up to 16 population doublings. These cells have raised interest as potential candidates for redifferentiation into functional insulin-producing cells. Previous work has associated dedifferentiation of cultured epithelial cells with epithelial-mesenchymal transition (EMT), and suggested that EMT generates cells with stem cell properties. Here we investigated the occurrence of EMT in these cultures and assessed their stem cell potential. METHODOLOGY/PRINCIPAL FINDINGS: Using cell-lineage tracing we provide direct evidence for occurrence of EMT in cells originating from beta cells in cultures of adult human islet cells. These cells express multiple mesenchymal markers, as well as markers associated with mesenchymal stem cells (MSC). However, we do not find evidence for the ability of such cells, nor of cells in these cultures derived from a non-beta-cell origin, to significantly differentiate into mesodermal cell types. CONCLUSIONS/SIGNIFICANCE: These findings constitute the first demonstration based on genetic lineage-tracing of EMT in cultured adult primary human cells, and show that EMT does not induce multipotency in cells derived from human beta cells

The influence of parathyroid hormone 1-34 on the osteogenic characteristics of adipose- and bone-marrow-derived mesenchymal stem cells from juvenile and ovarectomized rats

Mesenchymal stem cells (MSCs) are of growing interest in terms of bone regeneration. Most preclinical trials utilize bone-marrow-derived mesenchymal stem cells (bMSCs), although this is not without isolation and expansion difficulties. The aim of this study was: to compare the characteristics of bMSCs and adipose-derived mesenchymal stem cells (AdMSCs) from juvenile, adult, and ovarectomized (OVX) rats; and to assess the effect of human parathyroid hormone (hPTH) 1-34 on their osteogenic potential and migration to stromal cell-derived factor-1 (SDF-1)

Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES: Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days. METHODS: Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee. RESULTS: SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected. CONCLUSION: We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells

Cholangiocytes: Cell transplantation

Background:Due to significant limitations to the access to orthotropic liver transplantation, cell therapies forliver diseases have gained large interest worldwide.Scope of review:To revise current literature dealing with cell therapy for liver diseases. We discussed the ad-vantages and pitfalls of the different cell sources tested so far in clinical trials and the rationale underlying thepotential benefits of transplantation of human biliary tree stem cells (hBTSCs).Major conclusions:Transplantation of adult hepatocytes showed transient benefits but requires immune-sup-pression that is a major pitfall in patients with advanced liver diseases. Mesenchymal stem cells and hemato-poietic stem cells transplanted into patients with liver diseases are not able to replace resident hepatocytes butrather they target autoimmune or inflammatory processes into the liver. Stem cells isolated from fetal or adultliver have been recently proposed as alternative cell sources for advanced liver cirrhosis and metabolic liverdisease. We demonstrated the presence of multipotent cells expressing a variety of endodermal stem cell markersin (peri)-biliary glands of bile ducts in fetal or adult human tissues, and in crypts of gallbladder epithelium. Inthefirst cirrhotic patients treated in our center with biliary tree stem cell therapy, we registered no adverse eventbut significant benefits.General significance:The biliary tree stem cell could represent the ideal cell source for the cell therapy of liverdiseases. This article is part of a Special Issue entitled: Cholangiocytes in Health and Diseaseedited by JesusBanales, Marco Marzioni, Nicholas LaRusso and Peter Jansen

Transcriptionally and Functionally Distinct Mesenchymal Subpopulations Are Generated from Human Pluripotent Stem Cells

Summary: Various mesenchymal cell types have been identified as critical components of the hematopoietic stem/progenitor cell (HSPC) niche. Although several groups have described the generation of mesenchyme from human pluripotent stem cells (hPSCs), the capacity of such cells to support hematopoiesis has not been reported. Here, we demonstrate that distinct mesenchymal subpopulations co-emerge from mesoderm during hPSC differentiation. Despite co-expression of common mesenchymal markers (CD73, CD105, CD90, and PDGFRβ), a subset of cells defined as CD146hiCD73hi expressed genes associated with the HSPC niche and supported the maintenance of functional HSPCs ex vivo, while CD146loCD73lo cells supported differentiation. Stromal support of HSPCs was contact dependent and mediated in part through high JAG1 expression and low WNT signaling. Molecular profiling revealed significant transcriptional similarity between hPSC-derived CD146++ and primary human CD146++ perivascular cells. The derivation of functionally diverse types of mesenchyme from hPSCs opens potential avenues to model the HSPC niche and develop PSC-based therapies. : Crooks and colleagues demonstrated a previously underappreciated functional and molecular heterogeneity in mesenchyme generated from human pluripotent stem cells. Two mesenchymal subsets were distinguished by the reciprocal expression of CD146, CD73, and CD140a. CD146hiCD73hi mesenchyme supported self-renewing hematopoietic stem and progenitor cells (HSPCs), expressed markers of the HSPC niche, and shared a similar molecular signature with primary human adult pericytes. Keywords: pluripotent stem cell, mesenchyme, hematopoietic stem cell niche, pericyte biology, directed differentiation, mesoder

The Effect of Mechanical Cues on In Vitro Aging and Differentiation of Human Mesenchymal Stem Cells

Tissue engineering is a field that aims to replace or repair damaged tissue through the use of stem cells, biomaterials, and biomolecules. Human mesenchymal stem cells are multipotent adult stem cells that can be autologously transplanted. This work describes the effect of mechanical cues on human mesenchymal stem cells. An analysis on the age-related stiffening of these cells, and its effect on osteogenic and myogenic differentiation, is presented. This study gives insight to those using stem cells in vitro for extended periods of time. The effect of mechanical loading on stem cell differentiation is examined. Tensile and compressive loading are used to induce myogenic and osteogenic differentiation, respectively, in the absence of chemical cues. This study demonstrates that loading alone can accelerate differentiation. A 3-D cell culture method for cardiomyocyte differentiation is also explored. Numerous cardiomyocyte markers were observed, signifying that this method may be superior to chemical induction methods. A biodegradation study of four porous polymers is also presented, as scaffold choice is of great importance in the area of tissue engineering. This research provides guidance to those using human mesenchymal stem cells for tissue engineering