BFGF-selected Bone Marrow-derived Mesenchymal Stem Cells triggers the host response for bone regeneration
ABSTRACT: The classical concept of bone marrow-derived mesenchymal stem cells (BM-MSC), intended as a uniform, broad potent population, is progressively being substituted by the idea that the bone marrow harbors heterogeneous populations of non-hematopoietic stem cells. This in vivo heterogeneity is also amplified by the different experimental strategies used to isolate/culture them. Among the exogenous factors described to affect MSC in vitro growth, basic-fibroblast growth factor (bFGF) is one of the most common growth factors used to expand stem cells. Moreover, it has been reported that its signaling is associated with the mainteinance of stemness of a variety of stem cells, included MSC. Using an ectopic model of bone regeneration, we have previously described that the implantation of cells with different commitment levels, differentially influences the capacity to recruit host cells, activating endogenous regenerative mechanisms. Due to its properties, we here demonstrate that the addition of bFGF to primary BM cultures, leads to the selection of specific subpopulations able to induce a different host regenerative response, when in vivo implanted in association with suitable ceramic scaffolds. Moreover, taking advantage of a multiparametric and comparative genomic and proteomic approach, it has been evaluated how different culture conditions combine to bring about appreciable changes in the secretome of the cells, that consequently influence their in vivo regenerative behaviour. The full comprehension of the regulatory mechanisms that rule the host response depending on the type and differentiative stage of the transplanted cells could help us to develop novel clinical strategies where host cells could directly contribute to regenerate the appropriate tissue. Comparison of bFGF effects on bone marrow mesenchymal stem cells.
Project description:In animal models and human trials, intramyocardial injection of adult bone-marrow derived mesenchymal stem cells (BM-MSC) provides beneficial effects in failing hearts. These effects are mainly mediated through paracrine mechanisms. Mesenchymal stem cells of fetal origin (hAMC) can be isolated from the amniotic membrane of human placenta. Our results provide evidence that hAMC exert remarkable cardioprotective effects through paracrine mechanisms. However, the complete nature and scope of the paracrine mediators of cardioprotection have not been investigated yet. We compared the gene expression profiling of hAMC (n=8), BM-MSC (n=10) and dermal fibroblasts (n=6) to shed light onto the identity of putative cardioprotective factors secreted by fetal MSC. Total RNA was extracted from cultured hAMC (n=8), BM-MSC (n=10) and dermal fibroblasts (n=6) and analyzed with HumanHT-12 v3 Expression BeadChips
Project description:Mesenchymal stromal cells (MSC) are ideal candidates for cell therapies, due to their immune-regulatory and regenerative properties. We have previously reported that lung-derived MSC are tissue-resident cells with lung-specific properties compared to bone marrow-derived MSC. Assessing relevant molecular differences between lung-MSC and bone marrow-MSC is important, given that such differences may impact their behavior and potential therapeutic use. Here, we present an in-depth mass spectrometry (MS) based strategy to investigate the proteomes of lung-MSC and bone marrow-MSC. The MS-strategy relies on label free quantitative data-independent acquisition (DIA) analysis and targeted data analysis using a MSC specific spectral library. We identified several significantly differentially expressed proteins between lung-MSC and bone marrow-MSC within the cell layer (352 proteins) and in the conditioned medium (49 proteins). Bioinformatics analysis revealed differences in regulation of cell proliferation, which was functionally confirmed by decreasing proliferation rate through Cytochrome P450 stimulation. Our study reveals important tissue-specific differences within proteome and matrisome profiles between lung- and bone marrow-derived MSC that may influence their behavior and affect the clinical outcome when used for cell-therapy.
Project description:Transcription profiling analysis was performed on purified CD34+ cell lines (Cord Blood CD34+) treated with ExtracellularVescicles (EVs) isolated from bone marrow mesenchymal stem cells (BM-MSC).
Project description:Mesenchymal stem cells (MSC) are bone-marrow derived cells, capable of multipotent differentiation into connective tissues including bone, tendon and cartilage. They are an attractive source for autologous cell-based treatments for a range of clinical diseases and injuries. MSCs have been demonstrated to possess an age-related loss of cellular functions including differentiation potential and proliferation capacity; with implications for stem cell therapies in older patients. Furthermore the reduction in differentiation potential could contribute to ageing and age-related disease. Biological aging is coupled with a progressive reduction in the regulation of cellular, tissue and organ interaction, resulting in senescence. The purpose of this study was to investigate the epigenetic, RNA and protein changes in ageing MSCs in order to understand the age-related functional and biological changes required for their applications in regenerative medicine.
Project description:Autologous stem cell therapy has potential for biologic treatment of disc degeneration. Due to ease of harvest and abundance, adipose-derived mesenchymal stem cells (AD-MSC) are readily available. Our objectives were: 1) To develop/validate methods to harvest AD-MSC and direct them to a disc-like phenotype by three-dimensional (3D) culture and TGF-ß3 exposure; 2) To perform gene expression profiling for human AD-MSC and annulus cells in 3D culture; 3) To test whether disc cell-AD-MSC co-culture could augment proteoglycan production. Stem cell plasticity offers potential for future biologic therapies for disc degeneration. Data indicated that human AD-MSC can successfully be manipulated in 3D culture to express gene products important in the disc ECM (types I and II collagen, chondroitin sulfate, keratin sulfate, decorin), and that co-culture of annulus cells with AD-MSC enhances proteoglycan production. Studies defined gene expression patterns of AD-MSC and human annulus cells in 3D culture, important as we explore the potential of MSC in biologic therapies for disc degeneration. Experiment Overall Design: AD-MSC were extracted from human adipose tissue, and characterized as stem cells using accepted criteria (direction into osteoblasts or chondrocytes; and cell surface marker criteria). Three AD-MSC cultures were grown in 3D with or without TGF-ß3 for 2-3 weeks. Disc Tissue samples were obtained from surgical disc procedures performed on patients with herniated discs and degenerative disc disease. Seven disc cultures were also grown in 3-D for 2 weeks. RNA was harvested according to instructions with the Trizol isolation method, checked for quality using the 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA USA), reverse-transcribed to double-stranded cDNA, subjected to two rounds of transcription, and hybridized to the DNA microarray in the Affymetrix Fluidics Station 400. Affymetrix human U133 X3P arrays were used. Using Genesifter, gene expression in AD-MSC with and witout TGF-B3 was compared to annulus cells. Gene expression of stem cells with TGF-ß3 was also compared to stem cells grown in the absence of TGF-ß3.
Project description:Normal SJL mice, 6 to 8 weeks old, were used for the isolation of bone marrow stem cells (BMSC). Bone marrow cells were obtained from the femurs and tibias of euthanized mice by flushing with PBS. Cells were subjected to negative magnetic sorting using the Lineage Cell Depletion Kit. Isolation of murine bone marrow Lin-Sca-1+ cells was performed using MACS Sca-1 MultiSort Kit (fBMSC). The differentiation of neural stem cells was induced by removing the bFGF-containing medium and resuspending cells in fresh bFGF-free medium. Microarray analysis of miRNA expression profiles was performed comparing non differentiated bone marrow stem cells (fBMSC) to bone marrow stem cells differentiated for 4 or 7 days. The data are from adult mouse stem cells isolated from bone marrow
Project description:Regenerative medicine in aims to restore structure and function to tissues or organs damaged by time, disease or injury. Stem cells have great potential for tissue repair and regeneration, why they are intensely investigated in equine clinical research. However, before any type of stem cell can be applied in practice, it is crucial that the isolated stem cells have been definitively characterised by a set of specific functional or phenotypic markers. This project includes a surface mapping of equine mesenchymal (MSC) stem cell surface proteome.
Project description:Primary cortical neurons were isolated from E15 mice and after 5 days in vitro were untreated or treated for 24 h with mesenchymal stem cell conditioned medium and then untreated or treated for a further 24 h with NMDA. Neuron gene expression was profiled and compared between the four different conditions (neurons, neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA) to investigate the molecular mechanisms of MSC neuroprotection. Mesenchymal stem cells (MSC) promote functional recovery in experimental models of central nervous system (CNS) pathology and are currently being tested in clinical trials for stroke, multiple sclerosis and CNS injury. Their beneficial effects are attributed to activation of endogenous CNS repair processes and immune regulation but their mechanisms of action are poorly understood. Here we investigated the neuroprotective effects of MSC in simplified MSC-neuron co-culture systems and in mice using models of glutamate excitotoxicity. MSC protected primary cortical neurons against glutamate (NMDA) receptor-induced death and conditioned medium from MSC (MSC cm), but not control NIH3T3 cells, was sufficient for this effect. MSC cm neuroprotection in mouse cortical neurons was reduced by neutralizing antibodies to bFGF and associated with altered gene expression in neurons towards an immature phenotype as well as reduced neuronal Grin1, Grin2a and Grin2b mRNA levels in response to NMDA stimulation. Further, MSC cm neuroprotection in rat retinal ganglion cells was associated with absence of glutamate-induced calcium influx. Adoptive transfer of EGFP+MSC in a mouse kainic acid seizure model reduced CA3 neuron damage and hippocampal astrocytosis and resulted in the increased expression of neuronal genes that are upregulated by MSC cm, Bmi1, Ddx4, Ezh1, in the hippocampus. These results show that MSC mediate direct neuroprotection against glutamate excitotoxicity by secreting bFGF, reducing glutamate receptor expression and function and altering neuron gene expression towards an immature pattern, and provide evidence for a link between the therapeutic effects of MSC and the activation of endogenous repair processes following CNS injury. In vitro cultures primary cortical neurons from mice were protected from glutamate excitotoxicity when pre-treated with MSC cm. Global gene expression changes induced in neurons before and after treatment with MSC cm and/or NMDA were investigated using a cDNA spotted macroarray filter. Four samples were analysed in duplicate: neurons alone (untreated), neurons+MSC cm, neurons+NMDA, neurons+MSC cm+NMDA.
Project description:Schittler2010 - Cell fate of progenitor cells, osteoblasts or chondrocytes
Mathematical model describing the mechanism of differentiation of mesenchymal stem cells to bone (osteoblasts) or cartilage (chondrocytes) cells.
This model is described in the article:
Cell differentiation modeled via a coupled two-switch regulatory network.
Schittler D, Hasenauer J, Allgöwer F, Waldherr S.
Chaos 2010 Dec; 20(4): 045121
Mesenchymal stem cells can give rise to bone and other tissue cells, but their differentiation still escapes full control. In this paper we address this issue by mathematical modeling. We present a model for a genetic switch determining the cell fate of progenitor cells which can differentiate into osteoblasts (bone cells) or chondrocytes (cartilage cells). The model consists of two switch mechanisms and reproduces the experimentally observed three stable equilibrium states: a progenitor, an osteogenic, and a chondrogenic state. Conventionally, the loss of an intermediate (progenitor) state and the entailed attraction to one of two opposite (differentiated) states is modeled as a result of changing parameters. In our model in contrast, we achieve this by distributing the differentiation process to two functional switch parts acting in concert: one triggering differentiation and the other determining cell fate. Via stability and bifurcation analysis, we investigate the effects of biochemical stimuli associated with different system inputs. We employ our model to generate differentiation scenarios on the single cell as well as on the cell population level. The single cell scenarios allow to reconstruct the switching upon extrinsic signals, whereas the cell population scenarios provide a framework to identify the impact of intrinsic properties and the limiting factors for successful differentiation.
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