Osteocyte Lineages Research Articles

A Simple High Yield Technique for Isolation of Wharton's Jelly-derived Mesenchymal Stem Cell.

The isolation of Mesenchymal Stem Cells (MSCs) from various tissues is possible, with the umbilical cord emerging as a competitive alternative to bone marrow. In order to fulfill the demands of cell therapy, it is essential to generate stem cells on a clinical scale while minimizing time, cost, and contamination. Here is a simple and effective protocol for isolating MSC from Wharton's Jelly (WJ-MSC) using the explant method with various supplements. Utilizing the explant method, small fragments of Wharton's jelly from the human umbilical cord were cultured in a flask. The multipotency of the isolated cells, were confirmed by their differentiation ability to osteocyte and adipocyte. Additionally, the immunophenotyping of WJ-MSCs showed positive expression of CD73, CD90, and CD105, while remaining negative for hematopoietic markers CD34 and CD45, meeting the criteria for WJ-MSC identification. Following that, to evaluate cells' proliferative capacity, various supplements, including basic Fibroblast Growth Factor (bFGF), Non-Essential amino acids (NEA), and L-Glutamine (L-Gln) were added to either alpha-Minimal Essential Medium (α-MEM) or Dulbecco's Modified Eagle's Medium-F12 (DMEM-F12), as the basic culture media. WJ-MSCs isolated by the explant method were removed from the tissue after seven days and transferred to the culture medium. These cells differentiated into adipocyte and osteocyte lineages, expressing CD73, CD90, and CD105 positively and CD34 and CD45 negatively. The results revealed that addition of bFGF to α-MEM or DMEMF12 media significantly increased the proliferation of MSCs when compared to the control group. However, there were no significant differences observed when NEA or LGln were added. Although bFGF considerably enhances cell proliferation, our study demonstrates that MSCs can grow and expand when properly prepared Wharton's jelly tissues of the human umbilical cord.

MO280: Osteoblast-Specific Deletion of MEMO1 in Mice Severely Impairs the Expression of FGF23

Abstract BACKGROUND AND AIMS Memo deletion in mice caused a bone disease with diminished osteoblast and osteoclast biomarkers in serum [1], resembling the alterations occurring in adynamic bone disease in humans with renal failure, as well as those found in Klotho-deficient mice [2]. In addition, FGFR signaling is impaired in Memo-deficient osteoblasts [1]. We previously detected lower Fgf23 expression in cultured Memo-deficient osteoblasts, as the expression of FGF23 by bone cells requires autocrine or paracrine FGFR/Klotho signaling [3]. Here, we demonstrate that Memo1 in osteoblasts is required for Fgf23 expression in murine bone. METHOD Exon 2 of the Memo1 gene was deleted in the osteoblast–osteocyte lineage in Memo fl/fl mice using a Cre recombinase under the Col1a1 promotor to obtain osteoblast-specific knockout (obKO) mice. Memo obKO and Cre-negative littermate control mice were genotyped by tail genomic DNA. Organs were studied by microcomputed tomography, by qPCR and Western blot. Memo obKO and littermate control mice were challenged with acute kidney injury. RESULTS Memo obKO were viable without changes in gross anatomy. Weight at weaning age was transiently impaired in male but not in female Memo obKO compared with littermate controls (Fig. 1A). Memo1 expression was blunted in the femur of Memo obKO (Fig. 1B), whereas it remained comparable to controls in kidney (Fig. 1B) and liver tissues. Micro-CT revealed no differences between genotypes in trabecular or cortical bone of vertebrae, femur and tibia (Table 1). FGF23 gene expression by qPCR was undetectable in the femur of Memo obKO mice (Fig. 1C). Western blotting revealed a decrease of FGF23 protein in females and a tendency to lower expression in males of the Memo obKO genotype. CONCLUSION These findings demonstrate a role of Memo in the osteoblast–osteocyte lineage in contributing to the normal expression of FGF23. These data expand previous findings of FGFR-Klotho dependent regulation of FGF23 expression by the bone.

Mouse embryonic stem cells maintain differentiation potency into somatic lineage despite alternation of ploidy

Vertebrates, including mammals, are considered to have evolved by whole genome duplications. Although some fish have been reported to be polyploids that have undergone additional genome duplication, there have been no reports of polyploid mammals due to abnormal development after implantation. Furthermore, as the number of physiologically existing tetraploid somatic cells is small, details of the functions of these ploidy-altered cells are not fully understood. In this present study, we aimed to clarify the details of the differentiation potency of tetraploids using tetraploid embryonic stem cells. To clarify the differentiation potency, we used mouse tetraploid embryonic stem cells derived from tetraploid embryos. We presented tetraploid embryonic stem cells differentiated into neural and osteocyte lineage in vitro and tetraploid cells that contributed to various tissues of chimeric embryos ubiquitously in vivo. These results revealed that mouse embryonic stem cells maintain differentiation potency after altering the ploidy. Our results provide an important basis for the differentiation dynamics of germ layers in mammalian polyploid embryogenesis.

Immunomodulatory Effect of Human Umbilical Cord Blood-derived Mesenchymal Stem Cells on Activated T-lymphocyte.

Many studies have been performed about regenerative and immunomodulatory properties of mesenchymal stem cells (MSCs) and their application in different treatment approaches. The present study aimed to investigate the immunomodulatory effect of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) on the gene expression profile of cytokines in stimulated T-lymphocytes. For this purpose, MSCs were isolated from umbilical cord blood samples and cultured in Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. The nature of MSCs was identified by flow cytometry analysis and differentiation to the adipocyte and osteocyte lineage. Moreover, to investigate the immunomodulatory effects of MSCs on T cells, a co-culture system was designed and expression levels of interleukin (IL)-2, IL-4, IL-6, IL-10,IL-13, interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and transforming growth factor-beta (TGF-β) genes were measured; using the real-time polymerase chain reaction (RT-PCR) technique. Our results demonstrated the ability of MSCs to differentiate into adipocyte and osteocyte lineages. Further investigation also displayed that although UCB-MSCs could significantly reduce the expression of pro-inflammatory cytokines like IL-2, IL-6, IFN-γ, and TNF-α in activated T-lymphocytes, they noticeably potentiated the expression levels of IL-4, IL-10, IL-13, and TGF-β in the co-culture setting. In conclusion, UCB-MSCs have immunomodulatory effects on activated T-lymphocytes in favor of anti-inflammatory responses.

Does Physical Exercise Always Improve Bone Quality in Rats?

For decades, the osteogenic effect from different physical activities on bone in rodents remained uncertain. This literature review presents for the first time the effects on five exercise models (treadmill running, wheel running, swimming, resistance training and vibration modes) in three different experimental rat groups (males, females, osteopenic) on bone quality. The bone parameters presented are bone mineral density, micro-architectural and mechanical properties, and osteoblast/osteocyte and osteoclast parameters. This review shows that physical activities have a positive effect (65% of the results) on bone status, but we clearly observed a difference amongst the different protocols. Even if treadmill running is the most used protocol, the resistance training constitutes the first exercise model in term of osteogenic effects (87% of the whole results obtained on this model). The less osteogenic model is the vibration mode procedure (31%). It clearly appears that the gender plays a role on the bone response to swimming and wheel running exercises. Besides, we did not observe negative results in the osteopenic population with impact training, wheel running and vibration activities. Moreover, about osteoblast/osteocyte parameters, we conclude that high impact and resistance exercise (such jumps and tower climbing) seems to increase bone formation more than running or aerobic exercise. Among the different protocols, literature has shown that the treadmill running procedure mainly induces osteogenic effects on the viability of the osteocyte lineage in both males and females or ovariectomized rats; running in voluntary wheels contributes to a negative effect on bone metabolism in older male models; whole-body vertical vibration is not an osteogenic exercise in female and ovariectomized rats; whereas swimming provides controversial results in female models. For osteoclast parameters only, running in a voluntary wheel for old males, the treadmill running program at high intensity in ovariectomized rats, and the swimming program in a specific ovariectomy condition have detrimental consequences.

Comparison of the proteome patterns of adipose-derived stem cells with those treated with selegiline using a two dimensional gel electrophoresis analysis

ABSTRACTAdipose derived stem cells (ADSCs) are multipotent and can transdifferentiate into neural stem cells. We investigated the transdifferentiation of ADSCs to neural phenotype (NP) cells using selegiline and two-dimensional electrophoresis (2-DE). The perinephric and inguinal fat of rats was collected and used to isolate ADSCs that were characterized by immunophenotyping using flow cytometry. The ADSCs were differentiated into osteogenic and lipogenic cells. The NP cells were generated using 10−9 mM selegiline and characterized by immunocytochemical staining of nestin and neurofilament 68 (NF-68), and by qRT-PCR of nestin, neurod1 and NF68. Total protein of ADSCs and NP cells was extracted and their proteome patterns were examined using 2-DE. ADSCs carried CD73, CD44 and CD90 cell markers, but not CD34. ADSCs were differentiated into osteocyte and adipocyte lineages. The differentiated NP cells expressed nestin, neuro d1 and NF-68. The proteome pattern of ADSCs was compared with that of NP cells and eight spots showed more than a two fold increase in protein expression. The molecular weights and isoelectric points of these highly expressed proteins were estimated using Melanie software. We compared these results with those of the mouse proteomic database using the protein isoelectric point database, and the functions of the eight proteins in differentiation of NP cells were predicted using the UniProt database. The probable identities of the proteins that showed higher expression in NP cells included cholinesterase, GFRa2, protein kinase C (PKC-eta) and RING finger protein 121. The sequences of the proteins identified from mouse database were aligned by comparing them with similar proteins in rat database using the Basic Local Alignment Search Tool (BLAST). The E values of all aligned proteins were zero, which indicates consistency of the matched protein. These proteins participate in differentiation of the neuron and their overexpression causes ADSCs transdifferentiation into NP cells.

Potential Non-neoplastic Applications for Polyphenols in Stem Cell Utilization.

While polyphenols may have important effects on pluripotential stem cells that make them noteworthy as potential antineoplastic agents, their action on stem cells may portend other health benefits, such as treatments for cardiovascular and neurocognitive disorders. Resveratrol, the beststudied polyphenol, has been found to enable stem cells to differentiate into cardiomyocytes, neurons, osteocytes, and pancreatic beta cells, as well as facilitating augmentation of stem cell populations and protecting them from toxic injury. Curcumin protects mesenchymal stem cells from toxicity, and prevents them from facilitating chondrocytic hypertrophy. Quercetin enabled osteocytic and pancreatic beta cell differentiation, and protected neuronal stem cells from injury. Epigallocatechin gallate prevented damage to osteocyte precursors and averted differentiation into undesirable adipocytes. Genistein facilitated osteogenesis while preventing adipogenesis. Several other polyphenols, daidzein, caffeic and chlorogenic acid, kaempferol, and piceatannol, protect stems cells from reactive oxygen species and foster stem cells differentiation away from adipocytic and toward osteocytic lineages. Further research should better elucidate the pharmacokinetic profiles of each polyphenol, explore novel delivery systems, and expand investigation beyond rodent models to additional species.

Response of vascular mesenchymal stem/progenitor cells to hyperlipidemia.

Hyperlipidemia is a risk factor for atherosclerosis that is characterized by lipid accumulation, inflammatory cell infiltration, and smooth muscle cell proliferation. It is well known that hyperlipidemia is a stimulator for endothelial dysfunction and smooth muscle cell migration during vascular disease development. Recently, it was found that vessel wall contains a variable number of mesenchymal stem cells (MSCs) that are quiescent in physiological conditions, but can be activated by a variety of stimuli, e.g., increased lipid level or hyperlipidemia. Vascular MSCs displayed characteristics of stem cells which can differentiate into several types of cells, e.g., smooth muscle cells, adipocytic, chondrocytic, and osteocytic lineages. In vitro, lipid loading can induce MSC migration and chemokines secretion. After MSC migration into the intima, they play an essential role in inflammatory response and cell accumulation during the initiation and progression of atherosclerosis. In addition, MSC transplantation has been explored as a therapeutic approach to treat atherosclerosis in animal models. In this review, we aim to summarize current progress in characterizing the identity of vascular MSCs and to discuss the mechanisms involved in the response of vascular stem/progenitor cells to lipid loading, as well as to explore therapeutic strategies for vascular diseases and shed new light on regenerative medicine.

Expression of pluripotency markers in Arbas Cashmere goat hair follicle stem cells.

In our previous work, we found that the Inner Mongolia Arbas Cashmere goat hair follicle stem cells (gHFSCs) can be successfully differentiated into adipocyte, chondrocyte, and osteocyte lineages. In this study, we further examined the expression of the pluripotency and stemness markers Oct4, Nanog, Sox2, AKP, and TERT in gHFSCs by immunocytochemistry, flow cytometry, real-time PCR, and Western blot. Immunofluorescent staining showed that the gHFSCs were positive for all five markers. Fluorescence-activated cell sorting (FACS) further analyzed the positive expression of Oct4, Nanog, and Sox2 in the gHFSCs. Compared with Arbas Cashmere goat adipose-derived stem cells (gADSCs) at the mRNA expression level, Oct4 was relatively highly expressed in gHFSCs, 41.36 times of the gADSCs, and Nanog was 5.61, AKP was 2.74, and TERT was 2.10 times, respectively (p < 0.01). Western blot indicated that all markers are expressed at the protein level in the gHFSCs. When compared with gADSCs, using α-tubulin as a reference protein, gray intensity analysis showed that the expression of Oct4, Nanog, AKP, and TERT were, respectively, 5.94, 10.78, 1.33, and 1.39 times of gADSCs. Additionally, mRNA and protein expression of Sox2 were detected in the gHFSCs but not in the gADSCs. The protein expression pattern of these markers was consistent with the mRNA results.

Abstract 1408: Generation of 3D-microtissues suitable for drug screening with lentivirally GFP-labelled CD44+CD24- breast cancer cells enriched by irradiation

Abstract A stem cell like subpopulation of human T47D and MDA-MB-361 breast cancer cells (CD44+CD24-) was enriched by irradiation (2 Gy) and isolated using magnetic activated cell sorting. As an additional stem cell model, primary murine mesenchymal stem cells (MSCs) were isolated from the bone marrow of C3HxBL mice. We confirmed the stemness phenotype by repopulating heterogeneous cancer cell lines from isolated CD44+CD24- cells or by inducing differentiation of MSCs to the osteocyte, adipocyte and chondrocyte lineage. For gene delivery in cancer research, lentiviral transduction protocols are widely used though low transduction efficiencies of especially primary cells force the use of high lentiviral titers. In search for an optimized transduction protocol we came across a poloxamer-based chemical adjuvant to increase lentiviral gene delivery in primary cells [Höfig et al, Journal of Gene Medicine, 2012 and Höfig et al, Biomaterials, 2014]. Next we aimed to expand these benefits to transduce cells with a stem cell phenotype. After lentiviral transduction of a GFP encoding lentivirus in the presence of the poloxamer-based chemical adjuvant we could detect highest numbers of GFP expressing cells. Neither the chemical adjuvant nor the transgenic GFP expression altered the stemness phenotype of those cells analysed by repopulation or differentiation assays and by detection of stem cell markers on mRNA level (CD133 and Sox2). To monitor the fate of GFP-expressing CD44+CD24- cells, we cultured them in 3D-microtissues in mix with unlabelled bulk cells and analysed the effect of irradiation on cell number and location of CD44+CD24- cells within the spheroid. In this study we present a method for induction and isolation of human breast cancer cells with a stem cell like phenotype and developed an improved lentiviral transduction method by a chemical adjuvant preserving the stemness character in human and murine cells. Efficient lentiviral transduction enabled the generation of 3D-microtissues with GFP-labeled CD44+CD24- cells that both can be studied in cancer research and shall have applications for drug screening. Citation Format: Ines Höfig, Michael Rosemann, Michaela Albrecht, Yashodhara Ingawale, Jens M. Kelm, Michael J. Atkinson, Christian Thirion, Natasa Anastasov. Generation of 3D-microtissues suitable for drug screening with lentivirally GFP-labelled CD44+CD24- breast cancer cells enriched by irradiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1408. doi:10.1158/1538-7445.AM2015-1408

Mesenchymal Stem Cells from Pediatric Patients with Aplastic Anemia: Isolation, Characterization, Adipogenic, and Osteogenic Differentiation

Aplastic anemia is a syndrome of bone marrow (BM) failure characterized by peripheral pancytopenia and marrow hypoplasia. Its exact pathophysiology is still not clear. Mesenchymal stem cells (MSCs) play an important role in providing the specialized BM microenvironment for hematopoietic stem cells survival and differentiation. MSCs were isolated from BM of five patients with aplastic anemia and five controls. MSCs were characterized by morphology and immunophenotyping. Their viability, proliferative capacity, and adipogenic as well as osteogenic differentiation potentials were assessed. MSCs from aplastic anemia patients and controls shared similar spindle-shaped morphology and surface marker expression. MSCs derived from patients with aplastic anemia showed lower viability (74.2 ± 4.44% vs. 97.0 ± 1.58, p < 0.0001) and slower expansion rate as indicated by smaller population doubling and smaller cumulative population doubling from passages 1 to 4 (0.70 ± 0.22 vs. 2.34 ± 0.84; p = 0.009). Besides, aplastic anemia MSCs had poor capacity to differentiate into adipocytic and osteocytic lineages.

Tumor promotion through the mesenchymal stem cell compartment in human hepatocellular carcinoma

Although the infiltration of mesenchymal stem (stromal) cells (MSCs) into different tumors is widely recognized in animal models, the question whether these MSCs have a positive or negative effect on disease progression remains unanswered. The aim of this study is to investigate whether human hepatocellular carcinoma (HCC) harbors MSCs and whether these MSCs affect tumor growth. We observed that cells capable of differentiation into both adipocyte and osteocyte lineages and expressing MSC markers can be cultured from surgically resected HCC tissues. In situ staining of human HCC tissues with a STRO-1 antibody showed that the tumor and tumor-stromal region are significantly enriched with candidate MSCs compared with adjacent tissue (n = 12, P < 0.01). In mice, coengraftment of a human HCC cell line (Huh7) with MSCs resulted in substantially larger tumors compared with paired engraftment of Huh7 alone (n = 8, P < 0.01). Consistently, coculturing Huh7 with irradiated MSCs significantly increased the number and the size of colonies formed. This enhancement of Huh7 colony formation was also observed by treatment of MSC-conditioned medium (MSC-CM), suggesting that secreted trophic factors contribute to the growth-promoting effects. Genome-wide gene expression array and pathway analysis confirmed the upregulation of cell growth and proliferation-related processes and downregulation of cell death-related pathways by treatment of MSC-CM in Huh7 cells. In conclusion, these results show that MSCs are enriched in human HCC tumor compartment and could exert trophic effects on tumor cells. Thus, targeting of HCC tumor MSCs may represent a new avenue for therapeutic intervention.

Lamin A/C Haploinsufficiency Modulates the Differentiation Potential of Mouse Embryonic Stem Cells

BackgroundLamins are structural proteins that are the major determinants of nuclear architecture and play important roles in various nuclear functions including gene regulation and cell differentiation. Mutations in the human lamin A gene cause a spectrum of genetic diseases that affect specific tissues. Most available mouse models for laminopathies recapitulate disease symptoms for muscle diseases and progerias. However, loss of human lamin A/C also has highly deleterious effects on fetal development. Hence it is important to understand the impact of lamin A/C expression levels on embryonic differentiation pathways.Methodology and Principal FindingsWe have investigated the differentiation potential of mouse embryonic stem cells containing reduced levels of lamin A/C by detailed lineage analysis of embryoid bodies derived from these cells by in vitro culture. We initially carried out a targeted disruption of one allele of the mouse lamin A/C gene (Lmna). Undifferentiated wild-type and Lmna+/− embryonic stem cells showed similar expression of pluripotency markers and cell cycle profiles. Upon spontaneous differentiation into embryoid bodies, markers for visceral endoderm such as α-fetoprotein were highly upregulated in haploinsufficient cells. However, neuronal markers such as β-III tubulin and nestin were downregulated. Furthermore, we observed a reduction in the commitment of Lmna+/− cells into the myogenic lineage, but no discernible effects on cardiac, adipocyte or osteocyte lineages. In the next series of experiments, we derived embryonic stem cell clones expressing lamin A/C short hairpin RNA and examined their differentiation potential. These cells expressed pluripotency markers and, upon differentiation, the expression of lineage-specific markers was altered as observed with Lmna+/− embryonic stem cells.ConclusionsWe have observed significant effects on embryonic stem cell differentiation to visceral endoderm, neuronal and myogenic lineages upon depletion of lamin A/C. Hence our results implicate lamin A/C level as an important determinant of lineage-specific differentiation during embryonic development.

Glucagon-Like Peptide-1 Counteracts Oxidative Stress-Dependent Apoptosis of Human Cardiac Progenitor Cells by Inhibiting the Activation of the c-Jun N-terminal Protein Kinase Signaling Pathway

Increased apoptosis of cardiac progenitor cells (CPCs) has been proposed as a mechanism of myocardial damage and dysfunction. Glucagon-like peptide-1 (GLP-1) has been shown to improve heart recovery and function after ischemia and to promote cell survival. The protective effects of GLP-1 on oxidative stress-induced apoptosis were investigated in human CPCs isolated from human heart biopsies. Mesenchymal-type cells were isolated from human heart biopsies, exhibited the marker profile of CPCs, differentiated toward the myocardiocyte, adipocyte, chondrocyte, and osteocyte lineages under appropriate culture conditions, and expressed functional GLP-1 receptors. CPCs were incubated with GLP-1 with or without hydrogen peroxide (H(2)O(2)). Phospho- and total proteins were detected by immunoblotting and immunofluorescence analysis. Gene expression was evaluated by quantitative RT-PCR. The role of the canonical GLP-1 receptor was assessed by using the receptor antagonist exendin(9-39) and receptor-specific silencer small interfering RNAs. Cell apoptosis was quantified by an ELISA assay and by flow cytometry-detected Annexin V. Exposure of CPCs to H(2)O(2) induced a 2-fold increase in cell apoptosis, mediated by activation of the c-Jun N-terminal protein kinase (JNK) pathway. Preincubation of CPCs with GLP-1 avoided H(2)O(2)-triggered JNK phosphorylation and nuclear localization, and protected CPCs from apoptosis. The GLP-1 effects were markedly reduced by coincubation with the receptor antagonist exendin(9-39), small interfering RNA-mediated silencing of the GLP-1 receptor, and pretreatment with the protein kinase A inhibitor H89. In conclusion, activation of GLP-1 receptors prevents oxidative stress-mediated apoptosis in human CPCs by interfering with JNK activation and may represent an important mechanism for the cardioprotective effects of GLP-1.

Autologous Platelet-Rich Clot Releasate Stimulates Proliferation and Inhibits Differentiation of Adult Rat Tendon Stem Cells towards Nontenocyte Lineages

To explore the effects of autologous platelet-rich clot releasate (PRCR) on proliferation and differentiation of adult rat tendon stem cells (TSCs) in vitro, following intense mechanical stretching. TSCs were subjected to 8% mechanical stretching and subsequently incubated in control medium or medium supplemented with 2% or 10% PRCR. Collagen types I and III, peroxisome proliferator-activated receptor-γ (PPARγ), sex determining region Y-box 9 (SOX-9) and runt-related transcription factor 2 (RUNX2) concentrations were assessed via Western blotting and flow cytometry. Transforming growth factor (TGF)-β1 and vascular endothelial growth factor concentrations were measured using enzyme-linked immunosorbent assay. Treated TSCs were also cultured in adipogenic, chondrogenic or osteogenic culture media. PRCR increased the number of TSCs, and the concentrations of collagen types I and III and TGF-β1. In contrast, PRCR significantly reduced PPARγ, SOX-9 and RUNX2-positive cell numbers, and significantly reduced the numbers of TSC-derived adipocytes, chondrocytes and osteocytes. PRCR induced tenocyte differentiation while suppressing the adipocyte, chondrocyte and osteocyte lineages believed to impede tendon healing.

Mesenchymal stromal cell function is not affected by drugs used in the treatment of inflammatory bowel disease

Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I-III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays. The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-α on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied. MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-α antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment. This study demonstrates that, in vitro, MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-α therapy.

In vitro mesenchymal stem cell differentiation after mechanical stimulation

Mesenchymal stem cells (MSC) are multipotent cells capable of differentiating into adipocytic, chondrocytic and osteocytic lineages on suitable stimulation. We have hypothesized that mechanical loading may influence MSC differentiation and alter their phenotype accordingly. Mouse bone marrow-derived MSC were established in vitro by differential adherence to plastic culture plates and grown in low glucose medium with 10% foetal calf serum and growth factors. Cells grew out and were subcultured up to 20 times. Differentiation protocols were followed for several cell lineages. Clones with trilineage potential were seeded in type I collagen gels and incubated in a tensioning force bioreactor and real-time cell-derived forces were recorded. Gels were fixed and sectioned for light and electron microscopy. Cell monolayers of parent and cloned mouse bone marrow-derived MSC differentiated into adipocytes, osteocytes and chondrocytes, but not into cardiomyocytes, myotubes or neuronal cells. When cast into type I collagen gels and placed in tensioning bioreactors, MSC differentiated into fibroblast-like cells typical of tissue stroma, and upregulated α-smooth muscle actin, but rarely upregulated desmin. Electron microscopy showed collagen and elastin fibre synthesis into the matrix. These experiments confirmed that MSC cell fate choice depends on minute, cell-derived forces. Applied force could assist in commercial manufacture of cultured bio-engineered prostheses for regenerative medicine as it mimics tissue stresses and constitutes a good model for development of tissue substitutes.

Umbilical Cord Blood-Derived Mesenchymal Cell Fate After Mouse Umbilical Cord Blood Transplantation

Umbilical Cord Blood-Derived Mesenchymal Cell Fate After Mouse Umbilical Cord Blood Transplantation

Induction of an osteocyte-like phenotype by fibroblast growth factor-2

The purpose of this study was to characterize the molecular phenotype that occurs during the profound morphological shift of cultured osteogenic cells upon treatment with fibroblast growth factor-2 (FGF2). A time course of treatment with FGF2 was performed on an osteoblast cell line, primary bone marrow stromal cells and an osteocyte-like cell line. Morphologic changes were recorded, and gene profiling was carried out by real time PCR. By 8 h of FGF2 treatment, there is a striking morphological shift of osteoblast and stromal cells to an elongated dendritic-like morphology that is remindful of osteocytes. In osteoblasts treated with FGF2, this morphologic shift is preceded by an induction of several osteocyte markers, including dentin matrix protein 1 (>20-fold) and E11 (>5-fold). There is a transient increase in the gene expression of sclerostin (3.5-fold) and PHEX (2.5-fold). Sclerostin regulation by FGF2 is complex, as gene expression becomes markedly inhibited by FGF2 at times points after 8 h of treatment before rebounding at day 12. Analogous modulation of osteocyte markers is seen in bone marrow stromal cells and MLO-Y4 osteocyte-like cells. In conclusion, this study shows that FGF2 can regulate the transition of osteogenic cells towards the osteocyte lineage, as well as, regulate the expression of critical genes in osteocytes.

Stem Cells with Neural Crest Characteristics Derived from the Bulge Region of Cultured Human Hair Follicles

In this study, we demonstrate that we can isolate stem cells (SCs) with neural crest characteristics from the bulge area of cultured human hair follicles (HFs). These SCs can proliferate in situ and form spheroid structures attached to the bulge area of HFs, and they express immature neural crest cell markers but not differentiation markers. An expression profiling study showed that they share a similar gene expression pattern with murine skin immature neural crest cells. These human SCs are label-retaining cells and are capable of self-renewal through asymmetric cell division in vitro. They exhibit clonal multipotency that can give rise to myogenic, melanocytic, and neuronal cell lineages after in vitro clonal single cell culture. In addition, these SCs show differentiation potential toward mesenchymal lineages, and they can be differentiated into adipocyte, chondrocyte, and osteocyte lineages. Neuronal differentiation of these cells induces global gene expression changes with a significantly increased expression of neuron-associated genes. Differentiated neuronal cells can persist in mouse brain and retain neuronal differentiation markers. The presence of SCs with neural crest characteristics in HFs may offer new opportunities for the use of these cells in regenerative medicine.