Wnt signaling regulates the proliferation potential and lineage commitment of human umbilical cord derived mesenchymal stem cells

Relatively less is known about the interactions that tightly regulate the mesenchymal stem cells (MSCs) to maintain their pluripotency. Recent studies reports that Wnt proteins might play an important role in governing the MSC cell fate. In this study, we tested the hypothesis that Wnt proteins differentially regulate in vitro differentiation of human umbilical cord derived MSCs. Stromal cells from human umbilical cord (hUCMSCs) were isolated and treated with Wnt inhibitor/activator. FACS analysis of hUCMSCs for CD29, CD90, CD73, CD44, CD45 marker expression and gene expression of Wnt target genes and lineage specific genes were performed after Lithium Chloride (LiCl) and Quercetin treatment for 6 days. The cultured primary hUCMSCs demonstrated elevated MSC surface marker expression with clonogenic properties and differentiation potentials towards osteogenic, adipogenic and chondrogenic lineages. Downregulation in the expression of Wnt with Quercetin treatment was noted. LiCl treatment increased cellular proliferation but did not influence differentiation suggesting that the cells retain pluripotency whereas Quercetin treatment downregulated stemness markers, Wnt target gene expression and promoted osteogenesis as demonstrated by FACS analysis, calcium estimation and gene expression studies. Shift of differentiation potential after the inhibition of Wnt signaling by Quercetin was evident from the gene expression data and elevated calcium production, driving MSCs towards probable osteogenic lineage. The findings in particular are likely to open an interesting avenue of biomedical research, summarizing the impact of Wnt signaling on lineage commitment of MSCs.

     

Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.     

Impact of oxygen environment on mesenchymal stem cell expansion and chondrogenic differentiation

Introduction:  In vitro expansion and differentiation of mesenchymal stem cells (MSC) rely on specific environmental conditions, and investigations have demonstrated that one crucial factor is oxygen environment.
Objectives:  In order to understand the impact of oxygen tension on MSC culture and chondrogenic differentiation in vitro , we developed a mathematical model of these processes and applied it in predicting optimal assays.
Methods and results:  We compared ovine MSCs under physiologically low and atmospheric oxygen tension. Low oxygen tension improved their in vitro population growth as demonstrated by monoclonal expansion and colony forming assays. Moreover, it accelerated induction of the chondrogenic phenotype in subsequent three-dimensional differentiation cultures. We introduced a hybrid stochastic multiscale model of MSC organization in vitro . The model assumes that cell adaptation to non-physiological high oxygen tension reversibly changes the structure of MSC populations with respect to differentiation. In simulation series, we demonstrated that these changes profoundly affect chondrogenic potential of the populations. Our mathematical model provides a consistent explanation of our experimental findings.
Conclusions:  Our approach provides new insights into organization of MSC populations in vitro. The results suggest that MSC differentiation is largely reversible and that lineage plasticity is restricted to stem cells and early progenitors. The model predicts a significant impact of short-term low oxygen treatment on MSC differentiation and optimal chondrogenic differentiation at 10–11% pO₂.     

Differential roles of hypoxia inducible factor subunits in multipotential stromal cells under hypoxic condition

Cell therapy with bone marrow multipotential stromal cells (MSCs) represents a promising approach to promote wound healing and tissue regeneration. MSCs expanded in vitro lose early progenitors with differentiation and therapeutic potentials under normoxic condition, whereas hypoxic condition promotes MSC self-renewal through preserving colony forming early progenitors and maintaining undifferentiated phenotypes. Hypoxia inducible factor (HIF) pathway is a crucial signaling pathway activated in hypoxic condition. We evaluated the roles of HIFs in MSC differentiation, colony formation, and paracrine activity under hypoxic condition. Hypoxic condition reversibly decreased osteogenic and adipogenic differentiation. Decrease of osteogenic differentiation depended on HIF pathway; whereas decrease of adipogenic differentiation depended on the activation of unfolded protein response (UPR), but not HIFs. Hypoxia-mediated increase of MSC colony formation was not HIF-dependent also. Hypoxic exposure increased secretion of VEGF, HGF, and basic FGF in a HIF-dependent manner. These findings suggest that HIF has a limited, but pivotal role in enhancing MSC self-renewal and growth factor secretions under hypoxic condition.     

Dissolvable Gelatin-Based Microcarriers Generated through Droplet Microfluidics for Expansion and Culture of Mesenchymal Stromal Cells

Microcarriers are synthetic particles used in bioreactor-based cell manufacturing of anchorage-dependent cells to promote proliferation at efficient physical volumes, mainly by increasing the surface area-to-volume ratio. Mesenchymal stromal cells (MSCs) are adherent cells that are used for numerous clinical trials of autologous and allogeneic cell therapy, thus requiring avenues for large-scale cell production at efficiently low volumes and cost. Here, a dissolvable gelatin-based microcarrier is developed for MSC expansion. This novel microcarrier shows comparable cell attachment efficiency and proliferation rate when compared to several commercial microcarriers, but with higher harvesting yield due to the direct dissolution of microcarrier particles and thus reduced cell loss at the cell harvesting step. Furthermore, gene expression and in vitro differentiation suggest that MSCs cultured on gelatin microcarriers maintain trilineage differentiation with similar adipogenic differentiation efficiency and higher chondrogenic and osteogenic differentiation efficiency when compared to MSCs cultured on 2D planar polystyrene tissue culture flask; on the contrary, MSCs cultured on conventional microcarriers appear to be bipotent along osteochondral lineages whereby adipogenic differentiation potential is impeded. These results suggest that these gelatin microcarriers are suitable for MSC culture and expansion, and can also potentially be extended for other types of anchorage-dependent cells.     

Dissimilar differentiation of mesenchymal stem cells from bone marrow, umbilical cord blood, and adipose tissue

Mesenchymal stem cells (MSCs) have been investigated as promising candidates for use in new cell-based therapeutic strategies such as mesenchyme-derived tissue repair. MSCs are easily isolated from adult tissues and are not ethically restricted. MSC-related literature, however, is conflicting in relation to MSC differentiation potential and molecular markers. Here we compared MSCs isolated from bone marrow (BM), umbilical cord blood (UCB), and adipose tissue (AT). The isolation efficiency for both BM and AT was 100%, but that from UCB was only 30%. MSCs from these tissues are morphologically and immunophenotypically similar although their differentiation diverges. Differentiation to osteoblasts and chondroblasts was similar among MSCs from all sources, as analyzed by cytochemistry. Adipogenic differentiation showed that UCB-derived MSCs produced few and small lipid vacuoles in contrast to those of BM-derived MSCs and AT-derived stem cells (ADSCs) (arbitrary differentiation values of 245.57 +/- 943 and 243.89 +/- 145.52 mum(2) per nucleus, respectively). The mean area occupied by individual lipid droplets was 7.37 mum(2) for BM-derived MSCs and 2.36 mum(2) for ADSCs, a finding indicating more mature adipocytes in BM-derived MSCs than in treated cultures of ADSCs. We analyzed FAPB4, ALP, and type II collagen gene expression by quantitative polymerase chain reaction to confirm adipogenic, osteogenic, and chondrogenic differentiation, respectively. Results showed that all three sources presented a similar capacity for chondrogenic and osteogenic differentiation and they differed in their adipogenic potential. Therefore, it may be crucial to predetermine the most appropriate MSC source for future clinical applications.     

Neurogenic differentiation of mesenchymal stem cells: Transgenic approach

In addition to traditional osteogenic, chondrogenic, and adipogenic differentiation, mesenchymal stem cells are considered to be capable of also giving rise to neural lineage. We overview the transgenic approach for the neurogenic differentiation of MSCs, including the expression of neurotrophic factors, signaling molecules, and other transgenes with neurogenic properties.     

Multipotent mesenchymal stem cells from adult human eye conjunctiva stromal cells

Abstract Identification of mesenchymal stem cells (MSCs) derived from alternative sources has provided an exciting prospect for intensive investigation. This work focused on characterizing a new source of MSCs from stromal cells from human eye conjunctiva. In this study, after conjunctiva biopsies and culture of stromal segment of this tissue, fibroblast-like (SH2⁺, SH3⁺, CD29⁺, CD44⁺, CD166⁺, CD13⁺) human stromal cells, which can be differentiated toward the osteogenic, adipogenic, chondrogenic, and neurogenic lineages, were obtained. These cells expressed Oct-4, Nanog, Rex-1 genes, and some lineage-specific markers like cardiac actin and Keratin. Taken together, the results indicate that conjunctiva stromal-derived cells are a new source of multipotent MSCs and despite originating from an adult source, they express undifferentiated stem cell markers.     

Interleukin‐6 maintains bone marrow‐derived mesenchymal stem cell stemness by an ERK1/2‐dependent mechanism

Adult human mesenchymal stem cells (MSCs) hold promise for an increasing list of therapeutic uses due to their ease of isolation, expansion, and multi‐lineage differentiation potential. To maximize the clinical potential of MSCs, the underlying mechanisms by which MSC functionality is controlled must be understood. We have taken a deconstructive approach to understand the individual components in vitro, namely the role of candidate “stemness” genes. Our recent microarray gene expression profiling data suggest that interleukin‐6 (IL‐6) may contribute to the maintenance of MSCs in their undifferentiated state. In this study, we showed that IL‐6 gene expression is significantly higher in undifferentiated MSCs as compared to their chondrogenic, osteogenic, and adipogenic derivatives. Moreover, we found that MSCs secrete copious amounts of IL‐6 protein, which decreases dramatically during osteogenic differentiation. We further evaluated the role of IL‐6 for maintenance of MSC “stemness,” using a series of functional assays. The data showed that IL‐6 is both necessary and sufficient for enhanced MSC proliferation, protects MSCs from apoptosis, inhibits adipogenic and chondrogenic differentiation of MSCs, and increases the rate of in vitro wound healing of MSCs. We further identified ERK1/2 activation as the key pathway through which IL‐6 regulates both MSC proliferation and inhibition of differentiation. Taken together, these findings show for the first time that IL‐6 maintains the proliferative and undifferentiated state of bone marrow‐derived MSCs, an important parameter for the optimization of both in vitro and in vivo manipulation of MSCs. J. Cell. Biochem. 108: 577–588, 2009. Published 2009 Wiley‐Liss, Inc.     

sFRP2 Suppression of Bone Morphogenic Protein (BMP) and Wnt Signaling Mediates Mesenchymal Stem Cell (MSC) Self-renewal Promoting Engraftment and Myocardial Repair

Transplantation of mesenchymal stem cells (MSCs) is a promising therapy for ischemic injury; however, inadequate survival of implanted cells in host tissue is a substantial impediment in the progress of cellular therapy. Secreted Frizzled-related protein 2 (sFRP2) has recently been highlighted as a key mediator of MSC-driven myocardial and wound repair. Notably, sFRP2 mediates significant enhancement of MSC engraftment in vivo. We hypothesized that sFRP2 improves MSC engraftment by modulating self-renewal through increasing stem cell survival and by inhibiting differentiation. In previous studies we demonstrated that sFRP2-expressing MSCs exhibited an increased proliferation rate. In the current study, we show that sFRP2 also decreased MSC apoptosis and inhibited both osteogenic and chondrogenic lineage commitment. sFRP2 activity occurred through the inhibition of both Wnt and bone morphogenic protein (BMP) signaling pathways. sFRP2-mediated inhibition of BMP signaling, as assessed by levels of pSMAD 1/5/8, was independent of its effects on the Wnt pathway. We further hypothesized that sFRP2 inhibition of MSC lineage commitment may reduce heterotopic osteogenic differentiation within the injured myocardium, a reported adverse side effect. Indeed, we found that sFRP2-MSC-treated hearts and wound tissue had less ectopic calcification. This work provides important new insight into the mechanisms by which sFRP2 increases MSC self-renewal leading to superior tissue engraftment and enhanced wound healing.     

Arsenic trioxide promotes senescence and regulates the balance of adipogenic and osteogenic differentiation in human mesenchymal stem cells

Arsenic trioxide (ATO) as an anti-tumor drug could induce differentiation and apoptosis in tumor cells. Mesenchymal stem cells (MSCs) play important roles in the hematogenesis of bone marrow. Many reports have shown that the disorder of MSC adipogenic and osteogenic differentiation occurs in some diseases. However, reports about the effects of ATO on MSCs are limited. In this study, we found that 1 μM ATO promoted MSC senescence mainly through p21, although it had no effect on apoptosis at this dose. Furthermore, ATO promoted adipogenic differentiation, but inhibited osteogenic differentiation in MSCs. Our study also showed that CCAAT/enhancer-binding protein alpha C/EBPα and peroxisome proliferator-activated receptor gamma PPARγ might be involved in the regulation of adipogenic and osteogenic differentiation induced by ATO. Our results indicated that ATO may exert an anti-tumor effect by influencing bone marrow micro-environment. Moreover, it may regulate the adipogenic and osteogenic differentiation of MSCs.     

Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture

The in vitro culture of porcine bone marrow-derived mesenchymal stem cells (MSCs) was used for the investigation of adult stem cell biology. Isolated porcine MSCs possessed the ability to proliferate extensively in an antioxidants-rich medium containing 5% fetal bovine serum (FBS). Greater than 40 serial MSC passages and 100 cell population doublings have been recorded for some MSC batches. Early and late passage MSCs were defined here as those cultures receiving less than 5 trypsin passages and more than 15 trypsin passages, respectively. Consistent with their robust ability to proliferate, both the early and late passage MSCs expressed the cell-cycle promoting enzyme p34cdc2 kinase. Late MSCs, however, exhibited certain features reminiscent of cellular aging such as actin accumulation, reduced substrate adherence, and increased activity of lysosomal acid beta-galactosidase. Early MSCs retained the multipotentiality capable of chondrogenic, osteogenic, and adipogenic differentiation upon induction in vitro. In contrast, late MSCs were only capable of adipogenic differentiation, which was greatly enhanced at the expense of the osteochondrogenic potential. Along with these changes in multipotentiality, late MSCs expressed decreased levels of the bone morphogenic protein (BMP-7) and reduced activity of alkaline phosphatase. Late MSCs also exhibited attenuated synthesis of the hematopoietic cytokines granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), and stem cell factor (SCF). The long-term porcine MSC culture, thus, provides a model system to study the molecular interplay between multiple MSC differentiation cascades in the context of cellular aging.     

Clonal growth and differentiation of mesenchymal stromal cells from rat liver at different stages of embryogenesis

Fetal liver, during its hematopoietic activity, contains mesenchymal stromal cells (MSCs) generating its hematopoietic microenvironment. These cells are clonogenic and capable of multilineage differentiation; however, little is known about how their properties alter during embryogenesis. We compared the cloning efficiency of MSCs from rat fetal liver at 14, 16, and 20 days of development, as well as their capacity for osteo- and adipogenesis in vitro and chondrogenesis in vivo by ectopic transplantation of intact liver. The relative content of clonogenic MSCs in liver cell suspension was highest in 16-day fetuses and lowest in 20-day fetuses. Cells from 14-day fetuses exhibited high osteogenic and less apparent adipogenic and chondrogenic potential; cells from 20-day fetuses displayed weak adipogenic capacity and no osteo- or chondrogenic ability. These results show the correlation of MSC content and the cell differentiation potential with hematopoietic dynamics in developing rat liver. It may be thought that the changes we observed are related to the loss of hematopoietic activity and liver getting of definitive functions.     

Regulation of osteogenic and chondrogenic differentiation of mesenchymal stem cells in PEG-ECM hydrogels

Bone-marrow-derived mesenchymal stem cells (MSCs) are candidates for regeneration applications in musculoskeletal tissue such as cartilage and bone. Various soluble factors in the form of growth factors and cytokines have been widely studied for directing the chondrogenic and osteogenic differentiation of MSCs, but little is known about the way that the composition of extracellular matrix (ECM) components in three-dimensional microenvironments plays a role in regulating the differentiation of MSCs. To define whether ECM components influence the regulation of osteogenic and chondrogenic differentiation by MSCs, we encapsulated MSCs in poly-(ethylene glycol)-based (PEG-based) hydrogels containing exogenous type I collagen, type II collagen, or hyaluronic acids (HA) and cultured them for up to 6 weeks in chondrogenic medium containing transforming growth factor-β1 (10 ng/ml) or osteogenic medium. Actin cytoskeleton organization and cellular morphology were strongly dependent on which ECM components were added to the PEG-based hydrogels. Additionally, chondrogenic differentiation of MSCs was marginally enhanced in collagen-matrix-based hydrogels, whereas osteogenic differentiation, as measured by calcium accumulation, was induced in HA-containing hydrogels. Thus, the microenvironments created by exogenous ECM components seem to modulate the fate of MSC differentiation.