Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

The HIV proteins Tat and Nef promote human bone marrow mesenchymal stem cell senescence and alter osteoblastic differentiation

To maintain bone mass turnover and bone mineral density (BMD), bone marrow (BM) mesenchymal stem cells (MSCs) are constantly recruited and subsequently differentiated into osteoblasts. HIV‐infected patients present lower BMD than non‐HIV infected individuals and a higher prevalence of osteopenia/osteoporosis. In antiretroviral treatment (ART)‐naive patients, encoded HIV proteins represent pathogenic candidates. They are released by infected cells within BM and can impact on neighbouring cells. In this study, we tested whether HIV proteins Tat and/or Nef could induce senescence of human BM‐MSCs and reduce their capacity to differentiate into osteoblasts. When compared to nontreated cells, MSCs chronically treated with Tat and/or Nef up to 30 days reduced their proliferative activity and underwent early senescence, associated with increased oxidative stress and mitochondrial dysfunction. The antioxidant molecule N‐acetyl‐ cysteine had no or minimal effects on Tat‐ or Nef‐induced senescence. Tat but not Nef induced an early increase in NF‐κB activity and cytokine/chemokine secretion. Tat‐induced effects were prevented by the NF‐κB inhibitor parthenolide, indicating that Tat triggered senescence via NF‐κB activation leading to oxidative stress. Otherwise, Nef‐ but not Tat‐treated cells displayed early inhibition of autophagy. Rapamycin, an autophagy inducer, reversed Nef‐induced senescence and oxidative stress. Moreover, Tat+Nef had cumulative effects. Finally, Tat and/or Nef decreased the MSC potential of osteoblastic differentiation. In conclusion, our in vitro data show that Tat and Nef could reduce the number of available precursors by inducing MSC senescence, through either enhanced inflammation or reduced autophagy. These results offer new insights into the pathophysiological mechanisms of decreased BMD in HIV‐infected patients.     

Injection of synthetic mesenchymal stem cell mitigates osteoporosis in rats after ovariectomy

Osteoporosis is a severe skeletal disorder. Patients have a low bone mineral density and bone structural deterioration. Mounting lines of evidence suggest that inappropriate apoptosis of osteoblasts/osteocytes leads to maladaptive bone remodelling in osteoporosis. It has been suggested that transplantation of stem cells, including mesenchymal stem cells, may alter the trajectory of bone remoulding and mitigate osteoporosis in animal models. However, stem cells needed to be carefully stored and characterized before usage. In addition, there is great batch‐to‐batch variation in stem cell production. Here, we fabricated therapeutic polymer microparticles from the secretome and membranes of mesenchymal stem cells (MSCs). These synthetic MSCs contain growth factors secreted by MSCs. In addition, these particles display MSC surface molecules. In vitro, co‐culture with synthetic MSCs increases the viability of osteoblast cells. In a rat model of ovariectomy‐induced osteoporosis, injection of synthetic MSCs mitigated osteoporosis by reducing cell apoptosis and systemic inflammation, but increasing osteoblast numbers. Synthetic MSC offers a promising therapy to manage osteoporosis.     

Culture and differentiation of osteoblasts on coral scaffold from human bone marrow mesenchymal stem cells

In this paper we describe an approach that aims to provide fundamental information towards a scientific, biomechanical basis for the use of natural coral scaffolds to initiate mesenchymal stem cells into osteogenic differentiation for transplant purposes. Biomaterial, such as corals, is an osteoconductive material that can be used to home human derived stem cells for clinical regenerative purposes. In bone transplantation, the use of biomaterials may be a solution to bypass two main critical obstacles, the shortage of donor sites for autografts and the risk of rejection with allograft procedures. Bone regeneration is often needed for multiple clinical purposes for instance, in aesthetic reconstruction and regenerative procedures. Coral graft Porites lutea has been used by our team for a decade in clinical applications on over a thousand patients with different bone pathologies including spinal stenosis and mandibular reconstruction. It is well accepted that human bone marrow (hBM) is an exceptional source of mesenchymal stem cells (MSCs), which may differentiate into different cell phenotypes such as osteoblasts, chondrocytes, adipocytes, myocytes, cardiomyocytes and neurons. Isolated MSCs from human bone marrow were induced into osteoblasts using an osteogenic medium enriched with two specific growth factors, FGF9 and vitamin D2. Part of the cultured MSCs were directly transferred and seeded onto coral scaffolds (Porites Lutea) and induced to differentiate into osteoblasts and part were cultured in flasks for osteocell culture. The data support the concept that hBM is a reliable source of MSCs which may be easily differentiated into osteoblasts and seeded into coral as an optimal device for clinical application. Within this project we have also discussed the biological nature of MSCs, their potential application for clinical transplantation and the prospect of their use in gene therapy.     

Do microRNAs regulate bone marrow stem cell niche physiology?

The adult bone marrow, situated within the bone cavity, comprises three distinct stem cell populations: hematopoietic stem cells (HSCs), mesenchymal stromal/stem cells (MSCs) and endothelial progenitor/stem cells (EPCs). HSCs are a well-characterized population of self-renewing cells that give rise to all blood cells. The definition of MSCs is more complex due to the limited understanding of MSC properties. In general, MSCs are considered multipotent stromal cells that are able to differentiate into various cell types, including osteoblasts, chondrocytes and adipocytes. Compared to HSCs and MSCs, EPCs are a newly discovered population of stem/progenitor cells with the capacity to differentiate into endothelial cells, the cells forming the inner lining of a blood vessel.     

间充质干细胞在疾病治疗中的应用潜力

间充质干细胞（mesenchymal stem cell,MSCs）是衍生自中胚层的多能细胞,可产生多种间充质谱系,包括成骨细胞、脂肪细胞、成软骨细胞和肌细胞。MSCs还具有分泌多种细胞因子的能力,可促进血管生成、上皮再生等,在再生医学领域具有巨大的潜力。研究证实,MSCs可通过分化为多种细胞类型促进组织再生,加速伤口愈合;通过分泌细胞因子改善组织纤维化;还可通过携带载体药物诱导肿瘤细胞的凋亡,抑制肿瘤的发展。然而MSCs的成纤维化潜能和促进肿瘤生长的能力降低了MSCs应用于临床治疗的安全性。总结了MSCs在肿瘤、慢性难愈合伤口、纤维化等疾病发展过程中的作用,并进一步讨论了MSCs在临床相关疾病治疗中的潜在应用价值及挑战,以期为间充质干细胞的临床应用提供参考。     

The effect of quercetin on hepatic differentiation of human adipose-derived mesenchymal stem cells

Human adipose-derived mesenchymal stem cells (MSCs) can be stimulated to differentiate into hepatic cells. MSC differentiation was induced by fibroblast growth factor-4, hepatocyte growth factor, oncostatin M, and dexamethasone. The influence of quercetin on MSC hepatic differentiation in culture was assayed, and 1 or 10 μmole/L quercetin added into the induction medium enhanced the manifestation of MSC hepatic differentiation. Urea secretion, cytokeratin 19 expression, and α-fetoprotein synthesis were increased. Quercetin modulated CYP1A–cytochrome P450 activity in the differentiated cells. MSCs differentiated in the presence of quercetin exhibited higher viability and resistance to oxidative stress.     

Therapeutic Use of Stem Cell Transplantation for Cell Replacement or Cytoprotective Effect of Microvesicle Released from Mesenchymal Stem Cell

Idiopathic pulmonary fibrosis (IPF) is the most common and severe type of idiopathic interstitial pneumonias (IIP), and which is currently no method was developed to restore normal structure and function. There are several reports on therapeutic effects of adult stem cell transplantations in animal models of pulmonary fibrosis. However, little is known about how mesenchymal stem cell (MSC) can repair the IPF. In this study, we try to provide the evidence to show that transplanted mesenchymal stem cells directly replace fibrosis with normal lung cells using IPF model mice. As results, transplanted MSC successfully integrated and differentiated into type II lung cell which express surfactant protein. In the other hand, we examine the therapeutic effects of microvesicle treatment, which were released from mesenchymal stem cells. Though the therapeutic effects of MV treatment is less than that of MSC treatment, MV treat-ment meaningfully reduced the symptom of IPF, such as collagen deposition and inflammation. These data suggest that stem cell transplantation may be an effective strategy for the treatment of pulmonary fibrosis via replacement and cytoprotective effect of microvesicle released from MSCs.     

Differentiation of Rabbit Bone Mesenchymal Stem Cells into Endothelial Cells In Vitro and Promotion of Defective Bone Regeneration In Vivo

Tissue engineering strategies often fail to regenerate bones because of inadequate vascularization, especially in the reconstruction of large segmental bone defects. Large volumes of vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesis are difficult to obtain. In this study, we simulated bone healing by co-culturing differentiated ECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolic acid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairing rabbit mandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells. After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs. Immunohistochemical staining analysis with CD34 showed that the induced cells had the characteristics of ECs and MSC. The induced ECs were co-cultured in vitro, and the induction of MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR) staining experiments were performed, and the Coomassie brilliant blue total protein and ALP activity were measured. The MSCs proliferated and differentiated into osteoblast-like cells through direct contact between the derived ECs and MSCs. The co-cultured cells were seeded on PLGA scaffold to repair 1 cm mandible defects in the rabbit. The effectiveness of the repairs was assessed through soft X-ray and histological analyses. The main findings indicated that MSCs survived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. The results demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis and promoted new bone formation. This study may serve as a basis for the use of in vitro co-culturing techniques as an improvisation to bone tissue engineering for the repair of large bone defects.     

Study of differentiated human umbilical cord–derived mesenchymal stem cells transplantation on rat model of advanced parkinsonism

The aim of this study was to explore the curative effect of differentiated human umbilical cord–derived mesenchymal stem cells (hUC‐MSCs) transplantation on rat of advanced Parkinson disease (PD) model. Human umbilical cord–derived mesenchymal stem cells were cultured and induced differentiation in vitro. The PD rats were established and allocated randomly into 2 groups: differentiated hUC‐MSCs groups and physiological saline groups (the control group). Rotation test and immunofluorescence double staining were done. The result showed that hUC‐MSCs could differentiate into mature dopamine neurons. Frequency of rotation was significantly less in differentiated hUC‐MSCs groups than in normal saline group. After we transplanted these cells into the unilateral lesioned substantia nigra induced by striatal injection of 6‐hydroxydopamine and performed in the medial forebrain bundle and ventral tegmental area, nigral tyrosine hydroxylase–positive cells were observed and survival of at least 2 months. In addition, transplantation of hUC‐MSCs could make an obviously therapeutic effect on PD rats.     

Investigation of the mesenchymal stem cell compartment by means of a lentiviral barcode library

The hematopoietic bone marrow microenvironment is formed by proliferation and differentiation of mesenchymal stem cells (MSCs). The MSC compartment has been less studied than the hematopoietic stem cell compartment. To characterize the structure of the MSC compartment, it is necessary to trace the fate of distinct mesenchymal cells. To do so, mesenchymal progenitors need to be marked at the single-cell level. A method for individual marking of normal and cancer stem cells based on genetic “barcodes” has been developed for the last 10 years. Such approach has not yet been applied to MSCs. The aim of this study was to evaluate the possibility of using such barcoding strategy to mark MSCs and their descendants, colony-forming units of fibroblasts (CFU-Fs). Adherent cell layers (ACLs) of murine long-term bone marrow cultures (LTBMCs) were transduced with a lentiviral library with barcodes consisting of 32 + 3 degenerate nucleotides. Infected ACLs were suspended, and CFU-F-derived clones were obtained. DNA was isolated from each individual colony, and barcodes were analyzed in marked CFU-F-derived colonies by means of conventional polymerase chain reaction and Sanger sequencing. Barcodes were identified in 154 marked colonies. All barcodes appeared to be unique: there were no two distinct colonies bearing the same barcode. It was shown that ACLs included CFU-Fs with different proliferative potential. MSCs are located higher in the hierarchy of mesenchymal progenitors than CFU-Fs, so the presented data indicate that MSCs proliferate rarely in LTBMCs. A method of stable individual marking and comparing the markers in mesenchymal progenitor cells has been developed in this work. We show for the first time that a barcoded library of lentiviruses is an effective tool for studying stromal progenitor cells.     

A human iPSC model of Hutchinson Gilford Progeria reveals vascular smooth muscle and mesenchymal stem cell defects

The segmental premature aging disease Hutchinson-Gilford Progeria syndrome (HGPS) is caused by a truncated and farnesylated form of Lamin A called progerin. HGPS affects mesenchymal lineages, including the skeletal system, dermis, and vascular smooth muscle (VSMC). To understand the underlying molecular pathology of HGPS, we derived induced pluripotent stem cells (iPSCs) from HGPS dermal fibroblasts. The iPSCs were differentiated into neural progenitors, endothelial cells, fibroblasts, VSMCs, and mesenchymal stem cells (MSCs). Progerin levels were highest in MSCs, VSMCs, and fibroblasts, in that order, with these lineages displaying increased DNA damage, nuclear abnormalities, and HGPS-VSMC accumulating numerous calponin-staining inclusion bodies. Both HGPS-MSC and -VSMC viability was compromised by stress and hypoxia in vitro and in vivo (MSC). Because MSCs reside in low oxygen niches in vivo, we propose that, in HGPS, this causes additional depletion of the MSC pool responsible for replacing differentiated cells lost to progerin toxicity.     

Hepatogenic differentiation of mesenchymal stem cells induced by insulin like growth factor-I

AIM:To improve hepatic differentiation of human mesenchymal stem cell(MSC)using insulin growth factor 1(IGF-Ⅰ),which has important role in liver development,hepatocyte differentiation and function.METHODS:Bone marrow of healthy donors was aspirated from the iliac crest.The adherent cells expanded rapidly and were maintained with periodic passages until a relatively homogeneous population was established.The identification of these cells was carried out by immunophenotype analysis and differentiation potential into osteocytes and adipocytes.To effectively induce hepatic differentiation,we designed a protocol based on a combination of IGF-Ⅰ and liver specificfactors(hepatocyte growth factor,oncostatin M and dexamethasone).Morphological features,hepatic functions and cytological staining were assessed to evaluate transdifferentiation of human marrow-derived MSCs.RESULTS:Flow cytometric analysis and the differentiation potential into osteoblasts and adipocytes showed that more than 90% of human MSCs which were isolated and expanded were positive by specif ic markers and functional tests.Morphological assessment and evaluation of glycogen storage,albumin and α-feto protein expression,as well as albumin and urea secretion revealed a statistically signif icant difference between the experimental groups and control.CONCLUSION:In vitro differentiated MSCs using IGF-Ⅰwere able to display advanced liver metabolic functions,supporting the possibility of developing them as potential alternatives to primary hepatocytes.     

移植骨髓间充质干细胞治疗大鼠糖尿病的研究

目的 通过移植骨髓间充质干细胞（mesenchymal stem cell,MSC）的方法试治疗大鼠糖尿病。方法 贴壁生长的MSC与大鼠胰腺的细胞共培养以检测其向胰岛细胞分化的潜能。并将体外培养扩增的MSC移植入糖尿病大鼠体内,观测其能否改善糖尿病病情及其在大鼠体内微环境中的分化情况。结果 共培养法可使MSC分化为胰岛样细胞。对大鼠的MSC移植能明显缓解糖尿病病情。结论 MSC移植的方法对大鼠糖尿病有一定的治疗作用。     

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential

Background information. Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony‐forming ability and differentiation potential of four human cell types in vitro: commercially available skin‐derived fibroblasts [hSDFs (human skin‐derived fibroblasts)], adipose tissue‐derived stem cells [hASCs (human adipose tissue‐derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. Results. hSDFs, hASCs and WI38 exhibited a similar spindle‐like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell‐associated gene expressions by performing real‐time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5‐fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell‐derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. Conclusions. These findings suggest that (i) so‐called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony‐forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.     

Directing mesenchymal stem cells to bone to augment bone formation and increase bone mass

Aging reduces the number of mesenchymal stem cells (MSCs) that can differentiate into osteoblasts in the bone marrow, which leads to impairment of osteogenesis. However, if MSCs could be directed toward osteogenic differentiation, they could be a viable therapeutic option for bone regeneration. We have developed a method to direct MSCs to the bone surface by attaching a synthetic high-affinity and specific peptidomimetic ligand (LLP2A) against integrin α4β1 on the MSC surface to a bisphosphonate (alendronate, Ale) that has a high affinity for bone. LLP2A-Ale induced MSC migration and osteogenic differentiation in vitro. A single intravenous injection of LLP2A-Ale increased trabecular bone formation and bone mass in both xenotransplantation studies and in immunocompetent mice. Additionally, LLP2A-Ale prevented trabecular bone loss after peak bone acquisition was achieved or as a result of estrogen deficiency. These results provide proof of principle that LLP2A-Ale can direct MSCs to the bone to form new bone and increase bone strength.     

bFGF promotes adipocyte differentiation in human mesenchymal stem cells derived from embryonic stem cells

In this work we describe the establishment of mesenchymal stem cells (MSCs) derived from embryonic stem cells (ESCs) and the role of bFGF in adipocyte differentiation. The totipotency of ESCs and MSCs was assessed by immunofluorescence staining and RT-PCR of totipotency factors. MSCs were successfully used to induce osteoblasts, chondrocytes and adipocytes. MSCs that differentiated into adipocytes were stimulated with and without bFGF. The OD/DNA (optical density/content of total DNA) and expression levels of the specific adipocyte genes PPARγ2 (peroxisome proliferator activated receptor γ2) and C/EBPs were higher in bFGF cells. Embryonic bodies had a higher adipocyte level compared with cells cultured in plates. These findings indicate that bFGF promotes adipocyte differentiation. MSCs may be useful cells for seeding in tissue engineering and have enormous therapeutic potential for adipose tissue engineering.