Stem cell–related gene expression in clonal populations of mesenchymal stromal cells from bone marrow

Decline in the frequency of potent mesenchymal stem cells (MSCs) has been implicated in ageing and degenerative diseases. Increasing the circulating stem cell population can lead to renewed recruitment of these potent cells at sites of damage. Therefore, identifying the ideal cells for ex vivo expansion will form a major pursuit of clinical applications. This study is a follow-up of previous work that demonstrated the occurrence of fast-growing multipotential cells from the bone marrow samples. To investigate the molecular processes involved in the existence of such varying populations, gene expression studies were performed between fast- and slow-growing clonal populations to identify potential genetic markers associated with stemness using the quantitative real-time polymerase chain reaction comprising a series of 84 genes related to stem cell pathways. A group of 10 genes were commonly overrepresented in the fast-growing stem cell clones. These included genes that encode proteins involved in the maintenance of embryonic and neural stem cell renewal (sex-determining region Y-box 2, notch homolog 1, and delta-like 3), proteins associated with chondrogenesis (aggrecan and collagen 2 A1), growth factors (bone morphogenetic protein 2 and insulin-like growth factor 1), an endodermal organogenesis protein (forkhead box a2), and proteins associated with cell-fate specification (fibroblast growth factor 2 and cell division cycle 2). Expression of diverse differentiation genes in MSC clones suggests that these commonly expressed genes may confer the maintenance of multipotentiality and self-renewal of MSCs.

Cell sourcing for bone tissue engineering : amniotic fluid stem cells have a delayed, robust differentiation compared to mesenchymal stem cells

Cell based therapies for bone regeneration are an exciting emerging technology, but the availability of osteogenic cells is limited and an ideal cell source has not been identified. Amniotic fluid-derived stem (AFS) cells and bone-marrow derived mesenchymal stem cells (MSCs) were compared to determine their osteogenic differentiation capacity in both 2D and 3D environments. In 2D culture, the AFS cells produced more mineralized matrix but delayed peaks in osteogenic markers. Cells were also cultured on 3D scaffolds constructed of poly-e-caprolactone for 15 weeks. MSCs differentiated more quickly than AFS cells on 3D scaffolds, but mineralized matrix production slowed considerably after 5 weeks. In contrast, the rate of AFS cell mineralization continued to increase out to 15 weeks, at which time AFS constructs contained 5-fold more mineralized matrix than MSC constructs. Therefore, cell source should be taken into consideration when used for cell therapy, as the MSCs would be a good choice for immediate matrix production, but the AFS cells would continue robust mineralization for an extended period of time. This study demonstrates that stem cell source can dramatically influence the magnitude and rate of osteogenic differentiation in vitro.

Quantitative and qualitative assessment of the regenerative potential of osteoblasts versus bone marrow derived mesenchymal stem cells in the reconstruction of critical sized segmental tibial bone defects in a large animal model

This thesis is about the use of different cells for bone tissue engineering. The cells were used in combination with a novel biomaterial in a large tibial bone defects in a sheep model. Furthermore this study developed a novel cell delivery procedure for bone tissue engineering. This novel procedure of cell delivery could overcome the current problems of cell-based tissue engineering and serve as a baseline for the translation of novel concepts into clinical application.

Adipose differentiation of bone marrow-derived mesenchymal stem cells using Pluronic F-127 hydrogel in vitro

Due to increasing clinical demand for adipose tissue, a suitable scaffold for engineering adipose tissue constructs is needed. In this study, we have developed a three-dimensional (3-D) culture system using bone marrow-derived mesenchymal stem cells (BM-MSC) and a Pluronic F-127 hydrogel scaffold as a step towards the in vitro tissue engineering of fat. BM-MSC were dispersed into a Pluronic F-127 hydrogel with or without type I collagen added. The adipogenic differentiation of the BM-MSC was assessed by cellular morphology and further confirmed by Oil Red O staining. The BM-MSC differentiated into adipocytes in Pluronic F-127 in the presence of adipogenic stimuli over a period of 2 weeks, with some differentiation present even in absence of such stimuli. The addition of type I collagen to the Pluronic F-127 caused the BM-MSC to aggregate into clumps, thereby generating an uneven adipogenic response, which was not desirable.

Immunophenotypic, immunocytochemistry, ultrastructural, and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium-rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n=64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. © 2013 Wiley Periodicals, Inc.

Equine mesenchymal stem cells from bone marrow, adipose tissue and umbilical cord: immunophenotypic characterization and differentiation potential

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Equine peripheral blood-derived progenitors in comparison to bone marrow-derived mesenchymal stem cells

Fibroblast-like cells isolated from peripheral blood of human, canine, guinea pig, and rat have been demonstrated to possess the capacity to differentiate into several mesenchymal lineages. The aim of this work was to investigate the possibility of isolating pluripotent precursor cells from equine peripheral blood and compare them with equine bone marrow-derived mesenchymal stem cells. Human mesenchymal stem cells (MSCs) were used as a control for cell multipotency assessment. Venous blood (n = 33) and bone marrow (n = 5) were obtained from adult horses. Mononuclear cells were obtained by Ficoll gradient centrifugation and cultured in monolayer, and adherent fibroblast-like cells were tested for their differentiation potential. Chondrogenic differentiation was performed in serum-free medium in pellet cultures as a three-dimensional model, whereas osteogenic and adipogenic differentiation were induced in monolayer culture. Evidence for differentiation was made via biochemical, histological, and reverse transcription-polymerase chain reaction evaluations. Fibroblast-like cells were observed on day 10 in 12 out of 33 samples and were allowed to proliferate until confluence. Equine peripheral blood-derived cells had osteogenic and adipogenic differentiation capacities comparable to cells derived from bone marrow. Both cell types showed a limited capacity to produce lipid droplets compared to human MSCs. This result may be due to the assay conditions, which are established for human MSCs from bone marrow and may not be optimal for equine progenitor cells. Bone marrow-derived equine and human MSCs could be induced to develop cartilage, whereas equine peripheral blood progenitors did not show any capacity to produce cartilage at the histological level. In conclusion, equine peripheral blood-derived fibroblast-like cells can differentiate into distinct mesenchymal lineages but have less multipotency than bone marrow-derived MSCs under the conditions used in this study.

Bone marrow-derived mesenchymal stem cells become anti-angiogenic when chondrogenically or osteogenically differentiated:implications for bone and cartilage tissue engineering

Osteochondral tissue repair requires formation of vascularized bone and avascular cartilage. Mesenchymal stem cells stimulate angiogenesis both in vitro and in vivo but it is not known if these proangiogenic properties change as a result of chondrogenic or osteogenic differentiation. We investigated the angiogenic/antiangiogenic properties of equine bone marrow-derived mesenchymal stem cells (eBMSCs) before and after differentiation in vitro. Conditioned media from chondrogenic and osteogenic cell pellets and undifferentiated cells was applied to endothelial tube formation assays using Matrigel™. Additionally, the cell secretome was analysed using LC-MS/MS mass spectrometry and screened for angiogenesis and neurogenesis-related factors using protein arrays. Endothelial tube-like formation was supported by conditioned media from undifferentiated eBMSCs. Conversely, chondrogenic and osteogenic conditioned media was antiangiogenic as shown by significantly decreased length of endothelial tube-like structures and degree of branching compared to controls. Undifferentiated cells produced higher levels of angiogenesis-related proteins compared to chondrogenic and osteogenic pellets. In summary, eBMSCs produce an array of angiogenesis-related proteins and support angiogenesis in vitro via a paracrine mechanism. However, when these cells are differentiated chondrogenically or osteogenically, they produce a soluble factor(s) that inhibits angiogenesis. With respect to osteochondral tissue engineering, this may be beneficial for avascular articular cartilage formation but unfavourable for bone formation where a vascularized tissue is desired. © Copyright 2014, Mary Ann Liebert, Inc.

Sphingosine-1-phosphate mediates proliferation maintaining the multipotency of human adult bone marrow and adipose tissue-derived stem cells

High renewal and maintenance of multipotency of human adult stem cells (hSCs), are a prerequisite for experimental analysis as well as for potential clinical usages. The most widely used strategy for hSC culture and proliferation is using serum. However, serum is poorly defined and has a considerable degree of inter-batch variation, which makes it difficult for large-scale mesenchymal stem cells (MSCs) expansion in homogeneous culture conditions. Moreover, it is often observed that cells grown in serum-containing media spontaneously differentiate into unknown and/or undesired phenotypes. Another way of maintaining hSC development is using cytokines and/or tissue-specific growth factors; this is a very expensive approach and can lead to early unwanted differentiation. In order to circumvent these issues, we investigated the role of sphingosine-1-phosphate (S1P), in the growth and multipotency maintenance of human bone marrow and adipose tissue-derived MSCs. We show that S1P induces growth, and in combination with reduced serum, or with the growth factors FGF and platelet-derived growth factor-AB, S1P has an enhancing effect on growth. We also show that the MSCs cultured in S1P-supplemented media are able to maintain their differentiation potential for at least as long as that for cells grown in the usual serum-containing media. This is shown by the ability of cells grown in S1P-containing media to be able to undergo osteogenic as well as adipogenic differentiation. This is of interest, since S1P is a relatively inexpensive natural product, which can be obtained in homogeneous high-purity batches: this will minimize costs and potentially reduce the unwanted side effects observed with serum. Taken together, S1P is able to induce proliferation while maintaining the multipotency of different human stem cells, suggesting a potential for S1P in developing serum-free or serum-reduced defined medium for adult stem cell cultures.

Application of hyperbaric oxygen in bone tissue engineering : effect of hyperbaric oxygen treatment on bone marrow stem cells

and non-union of bony fractures has been proposed since 1966, little has been known about the effect of HBOT on bone marrow stem cells (BMSC). The aim of this study is to investigate the effect of HBO treatment on osteogenetic differentiation of BMSC and potential application in bone tissue engineering. Adhesive stromal cells harvested from bone marrow were characterized by mesenchymal differentiation potential, cell surface markers and their proliferation capacity. Mesenchymal stem cells, which demonstrated osteogenic, chondrogenic and adipogenic differentiation potential and expressed positively for CD 29, CD 44, CD 73, CD 90, CD 105, CD 166 and negatively for CD34 and CD 45, were selected and treated in a laboratory-scale HBO chamber using different oxygen pressures and exposure times. No obvious effect of HBO treatment on BMSC proliferation was noticed. However, cytotoxic effects of HBO were considerably less pronounced when cells were cultured in medium supplemented with 10% FBS in comparison to medium supplemented with 2% FCS, as was evaluated by WST-1 assay. Under HBO treatment, bone nodules were formed in three days, which was clearly revealed by Von Kossa staining. In contrasts, without HBO treatment, bone nodules were not detected until 9-12 days using the same inducing culture media. Calcium deposition was also significantly increased after three days of HBO treatments compared to no HBO treatment. In addition it was also found that oxygen played a direct role in the enhancement of BMSC osteogenic differentiation, which was independent of the effect of air pressure.

Neural differentiation potential of human bone marrow-derived mesenchymal stromal cells: misleading marker gene expression

Background In contrast to pluripotent embryonic stem cells, adult stem cells have been considered to be multipotent, being somewhat more restricted in their differentiation capacity and only giving rise to cell types related to their tissue of origin. Several studies, however, have reported that bone marrow-derived mesenchymal stromal cells (MSCs) are capable of transdifferentiating to neural cell types, effectively crossing normal lineage restriction boundaries. Such reports have been based on the detection of neural-related proteins by the differentiated MSCs. In order to assess the potential of human adult MSCs to undergo true differentiation to a neural lineage and to determine the degree of homogeneity between donor samples, we have used RT-PCR and immunocytochemistry to investigate the basal expression of a range of neural related mRNAs and proteins in populations of non-differentiated MSCs obtained from 4 donors. Results The expression analysis revealed that several of the commonly used marker genes from other studies like nestin, Enolase2 and microtubule associated protein 1b (MAP1b) are already expressed by undifferentiated human MSCs. Furthermore, mRNA for some of the neural-related transcription factors, e.g. Engrailed-1 and Nurr1 were also strongly expressed. However, several other neural-related mRNAs (e.g. DRD2, enolase2, NFL and MBP) could be identified, but not in all donor samples. Similarly, synaptic vesicle-related mRNA, STX1A could only be detected in 2 of the 4 undifferentiated donor hMSC samples. More significantly, each donor sample revealed a unique expression pattern, demonstrating a significant variation of marker expression. Conclusion The present study highlights the existence of an inter-donor variability of expression of neural-related markers in human MSC samples that has not previously been described. This donor-related heterogeneity might influence the reproducibility of transdifferentiation protocols as well as contributing to the ongoing controversy about differentiation capacities of MSCs. Therefore, further studies need to consider the differences between donor samples prior to any treatment as well as the possibility of harvesting donor cells that may be inappropriate for transplantation strategies.