Adult Bone Marrow Stromal Cells Research Articles

Derivation of Oligodendrocyte Precursors from Adult Bone Marrow Stromal Cells for Remyelination Therapy.

Transplantation of oligodendrocyte precursors (OPs) is potentially therapeutic for myelin disorders but a safe and accessible cell source remains to be identified. Here we report a two-step protocol for derivation of highly enriched populations of OPs from bone marrow stromal cells of young adult rats (aMSCs). Neural progenitors among the aMSCs were expanded in non-adherent sphere-forming cultures and subsequently directed along the OP lineage with the use of glial-inducing growth factors. Immunocytochemical and flow cytometric analyses of these cells confirmed OP-like expression of Olig2, PDGFRα, NG2, and Sox10. OPs so derived formed compact myelin both in vitro, as in co-culture with purified neurons, and in vivo, following transplantation into the corpus callosum of neonatal shiverer mice. Not only did the density of myelinated axons in the corpus callosum of recipient shiverer mice reach levels comparable to those in age-matched wild-type mice, but the mean lifespan of recipient shiverer mice also far exceeded those of non-recipient shiverer mice. Our results thus promise progress in harnessing the OP-generating potential of aMSCs towards cell therapy for myelin disorders.

Modeling adult skeletal stem cell response to laser-machined topographies through deep learning

The response of adult human bone marrow stromal stem cells to surface topographies generated through femtosecond laser machining can be predicted by a deep neural network. The network is capable of predicting cell response to a statistically significant level, including positioning predictions with a probability P < 0.001, and therefore can be used as a model to determine the minimum line separation required for cell alignment, with implications for tissue structure development and tissue engineering. The application of a deep neural network, as a model, reduces the amount of experimental cell culture required to develop an enhanced understanding of cell behavior to topographical cues and, critically, provides rapid prediction of the effects of novel surface structures on tissue fabrication and cell signaling.

C-KIT Expression Distinguishes Fetal from Postnatal Skeletal Progenitors.

SummaryHematopoietic stem cells (HSCs) and skeletal stem cells (SSCs) cohabit in the bone marrow. KITL (C-KIT ligand) from LEPR+ adult bone marrow stromal cells is pivotal for HSC maintenance. In contrast, it remains unclear whether KITL/C-KIT signaling also regulates SSCs. Here, we lineage traced C-KIT+ cells and found that C-KIT was expressed by fetal, but not postnatal skeletal progenitors. Fetal C-KIT+ cells gave rise to 20% of LEPR+ stromal cells in adult bone marrow, forming nearly half of all osteoblasts. Disruption of mTOR signaling in fetal C-KIT+ cells impaired bone formation. Notably, conditional deletion of Kitl from PRX1+ fetal bone marrow stromal cells, but not LEPR+ adult bone marrow stromal cells, significantly increased bone formation. Thus, our work identified C-KIT+ skeletal progenitors as an important source of bones formed during development.

Lack of a skeletal muscle phenotype in adult human bone marrow stromal cells following xenogeneic-free expansion

BackgroundMany studies have elegantly shown that murine and rat bone marrow-derived mesenchymal stromal cells (bmMSCs) contribute to muscle regeneration and improve muscle function. Yet, the ability of transplanted human bmMSCs to manifest myogenic potential shows conflicting results. While human adipose- and umbilical cord-derived MSCs can be differentiated into a skeletal muscle phenotype using horse serum (HS), bmMSCs have only been shown to differentiate towards the skeletal muscle lineage using a complex mixture of cytokines followed by transfection with notch intracellular domain.MethodsSince xenogeneic-free growth supplements are increasingly being used in the expansion of bmMSCs in clinical trials, we investigated the effects of human plasma and platelet lysate (P/PL) on the expression of neuromuscular markers and whether P/PL-expanded human bmMSCs could be differentiated towards a skeletal myogenic phenotype. Neuromuscular markers were measured using the highly sensitive droplet digital polymerase chain reaction for measuring the expression of Myf5, MyoD, MyoG, ACTA1, Desmin, GAP-43, and Coronin 1b transcripts, by performing immunofluorescence for the expression of Desmin, GAP-43, and MEF2, and flow cytometry for the expression of CD56/neural cell adhesion molecule (NCAM).ResultsDespite that bmMSCs expressed the myogenic regulatory factor (MRF) MEF2 after expansion in P/PL, bmMSCs cultured under such conditions did not express other essential MRFs including Myf5, MyoD, MyoG, or ACTA1 needed for myogenesis. Moreover, HS did not induce myogenesis of bmMSCs and hence did not induce the expression of any of these myogenic markers. P/PL, however, did lead to a significant increase in neurogenic GAP-43, as well as Desmin expression, and resulted in a high baseline expression of the neurogenic gene Coronin 1b which was sustained under further P/PL or HS culture conditions. Fetal bovine serum resulted in equally high levels of GAP-43 and Coronin 1b. Moreover, the proportion of CD56/NCAM-positive bmMSCs cultured in P/PL was 5.9 ± 2.1.ConclusionsThese data suggest that P/PL may prime a small portion of bmMSCs towards an early neural precursor cell type. Collectively, this shows that P/PL partially primes the cells towards a neurogenic phenotype, but does not prime adult human bmMSCs towards the skeletal muscle lineage.

Identification of neurospheres generated from human dental pulp stem cells in xeno-/serum-free conditions

IntroductionCell-based therapies require an emerging alternative treatment using easily harvested cell sources. Neural stem cells derived from various tissues, including brain, bone marrow, skin and retina can give rise to both neurons and glial cells. Recently, human dental pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHED) were demonstrated to have mesenchymal stem cell-like abilities such as self-renewal and multi-lineage differentiation, including neuron and glial cells. Moreover, DPSCs and SHED show a higher proliferation rate and a higher number of population doublings compared with adult bone marrow stromal stem cells. Therefore, DPSCs are a useful source that can be applied in cell replacement therapy for various neurological disorders. Generally, the conventional culture methods for DPSCs have used serum, therefore the undefined components in culture medium may complicate investigations of the molecular mechanisms that control the self-renewal and differentiation of DPSCs. However, neural stem cells proliferate to form ‘neurospheres’ in suspension in vitro in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF). No study to date has obtained neurospheres from DPSCs in serum-free conditions in primary culture. Thus, the aim of this study was to establish a method for the proliferation and neural differentiation of DPSCs in xeno- and serum-free conditions in primary culture.MethodsDPSCs were obtained from the dental pulp of wisdom teeth from healthy individuals (18–41 years old) and cultured in conventional medium containing 15% fetal bovine serum and xeno-/serum-free medium. We evaluated the proliferation of DPSCs, neurosphere generation, and neural differentiation under xeno-/serum-free conditions by flow cytometry, immunohistochemistry, and real-time polymerase chain reaction.ResultsIn proliferation medium without xeno/serum, DPSCs can proliferate and generate neurospheres, however, the neurospheres had limited self-renewal ability. Under differentiation conditions, class III β-tubulin (TUBB3) and microtubule-associated protein (MAP2) were more significantly expressed in neurospheres derived from DPSCs in xeno-/serum-free culture conditions than in DPSCs in conventional culture conditions.ConclusionsOur result demonstrated that neurosphere generation from DPSCs in xeno-/serum-free culture may be an accessible source for clinical cell replacement therapies for neuronal degenerative diseases.

Osteogenic potential of adipogenic predifferentiated human bone marrow-derived multipotent stromal cells for bone tissue-engineering.

In the present study, we evaluated the benefits of an adipogenic predifferentiation, the pathway most closely related to osteoblastogenesis, on the pro-osteogenic potential of human adult multipotent bone marrow stromal cells (hBMSCs), both in vitro and in vivo. Adipogenic differentiation of hBMSCs for 14days resulted in a heterogeneous cell population from which the most adipogenic-committed cells were eliminated by their lack of readhesion ability. Our results provided evidence that the select adherent adipogenic differentiated hBMSCs (sAD+ cells) express a gene profile characteristic of both adipogenic and osteogenic lineages. In vitro, when cultured in osteogenic medium, sAD+ differentiated along the osteogenic lineage faster than undifferentiated hBMSCs. In vivo, in an ectopic mouse model, sAD+ exhibited a significantly higher bone formation capability compared with undifferentiated hBMSCs. We sought, then, to investigate the underlying mechanisms responsible for such beneficial effects of adipogenic predifferentiation on bone formation and found that this outcome was not linked to a better cell survival post-implantation. The secretome of sAD+ was both proangiogenic and chemoattractant, but its potential did not supersede the one of undifferentiated hBMSCs. However, using co-culture systems, we observed that the sAD+ paracrine factors were pro-osteogenic on undifferentiated hBMSCs. In conclusion, adipogenic priming endows hBMSCs with high osteogenic potential as well as pro-osteogenic paracrine-mediated activity. This preconditioning appears as a promising strategy for bone tissue engineering technology in order to improve the hBMSC osteogenic potency in vivo.

Chromosome copy number variation in telomerized human bone marrow stromal cells; insights for monitoring safe ex-vivo expansion of adult stem cells

Adult human bone marrow stromal cells (hBMSC) cultured for cell therapy require evaluation of potency and stability for safe use. Chromosomal aberrations upsetting genomic integrity in such cells have been contrastingly described as “Limited” or “Significant”. Previously reported stepwise acquisition of a spontaneous neoplastic phenotype during three-year continuous culture of telomerized cells (hBMSC-TERT20) didn't alter a diploid karyotype measured by spectral karyotype analysis (SKY). Such screening may not adequately monitor abnormal and potentially tumorigenic hBMSC in clinical scenarios. We here used array comparative genomic hybridization (aCGH) to more stringently compare non-tumorigenic parental hBMSC-TERT strains with their tumorigenic subcloned populations. Confirmation of a known chromosome 9p21 microdeletion at locus CDKN2A/B, showed it also impinged upon the adjacent MTAP gene. Compared to reference diploid human fibroblast genomic DNA, the non-tumorigenic hBMSC-TERT4 cells had a copy number variation (CNV) in at least 14 independent loci. The pre-tumorigenic hBMSC-TERT20 cell strain had further CNV including 1q44 gain enhancing SMYD3 expression and 11q13.1 loss downregulating MUS81 expression. Bioinformatic analysis of gene products reflecting 11p15.5 CNV gain in tumorigenic hBMSC-TERT20 cells highlighted networks implicated in tumorigenic progression involving cell cycle control and mis-match repair. We provide novel biomarkers for prospective risk assessment of expanded stem cell cultures.

Directed Differentiation of Human Bone Marrow Stromal Cells to Fate-Committed Schwann Cells.

SummaryOur ultimate goal of in vitro derivation of Schwann cells (SCs) from adult bone marrow stromal cells (BMSCs) is such that they may be used autologously to assist post-traumatic nerve regeneration. Existing protocols for derivation of SC-like cells from BMSCs fall short in the stability of the acquired phenotype and the functional capacity to myelinate axons. Our experiments indicated that neuro-ectodermal progenitor cells among the human hBMSCs could be selectively expanded and then induced to differentiate into SC-like cells. Co-culture of the SC-like cells with embryonic dorsal root ganglion neurons facilitated contact-mediated signaling that accomplished the switch to fate-committed SCs. Microarray analysis and in vitro myelination provided evidence that the human BMSC-derived SCs were functionally mature. This was reinforced by repair and myelination phenotypes observable in vivo with the derived SCs seeded into a nerve guide as an implant across a critical gap in a rat model of sciatic nerve injury.

Mechanical induction of dentin-like differentiation by adult mouse bone marrow stromal cells using compressive scaffolds

Tooth formation during embryogenesis is controlled through a complex interplay between mechanical and chemical cues. We have previously shown that physical cell compaction of dental mesenchyme cells during mesenchymal condensation is responsible for triggering odontogenic differentiation during embryogenesis, and that expression of Collagen VI stabilizes this induction. In addition, we have shown that synthetic polymer scaffolds that artificially induce cell compaction can induce embryonic mandible mesenchymal cells to initiate tooth differentiation both in vitro and in vivo. As embryonic cells would be difficult to use for regenerative medicine applications, here we explored whether compressive scaffolds coated with Collagen VI can be used to induce adult bone marrow stromal cells (BMSCs) to undergo an odontogenic lineage switch. These studies revealed that when mouse BMSCs are compressed using these scaffolds they increase expression of critical markers of tooth differentiation in vitro, including the key transcription factors Pax9 and Msx1. Implantation under the kidney capsule of contracting scaffolds bearing these cells in mice also resulted in local mineralization, calcification and production of dentin-like tissue. These findings show that these chemically-primed compressive scaffolds can be used to induce adult BMSCs to undergo a lineage switch and begin to form dentin-like tissue, thus raising the possibility of using adult BMSCs for future tooth regeneration applications.

Angiogenic potency evaluation of cell therapy candidates by a novel application of the in vitro aortic ring assay

BackgroundDue to limitations of current angiogenesis assays, we aimed to develop a novel application of the rat aortic ring assay to assess the angiogenic potential of mesenchymal stromal cells (MSCs). First-trimester human umbilical cord-derived perivascular cells (FTM HUCPVCs) have multipotent characteristics and previously demonstrated angiogenic potential. We compared the effect of this young source of MSCs and adult bone marrow stromal cells (BMSCs) on ex vivo aortic endothelial network formation.MethodsThoracic segments of adult rat aortas were isolated, sectioned and embedded into Matrigel™. Fluorophore-labeled FTM HUCPVC lines and BMSCs (N = 3) were cocultured with developing endothelial networks (day 0). MSC integration, tube formation and endothelial network growth were monitored daily using phase-contrast and fluorescence microscopy. Quantification of endothelial networks was performed using ImageJ network analysis software on day 5 of coculture.ResultsFTM HUCPVCs from two umbilical cord samples migrated toward and integrated with developing aortic ring tubular networks while displaying elongated morphologies (day 1). In contrast, BMSCs did not show targeted migration and maintained spherical morphologies with limited physical interactions. Within 1 week of coculture, FTM HUCPVC lines contributed to significantly greater radial network growth and network loop formation when compared to BMSCs and untreated networks.ConclusionsWe have developed a novel potency assay to assess the angiogenic potential of cell therapy candidates. Favorable properties of FTM HUCPVCs over BMSCs that we observed with this assay and which merit further study include chemotaxis, affinity for developing vasculature, and physical supportive interactions contributing to the development of endothelial networks.

Rapid and efficient generation of neural progenitors from adult bone marrow stromal cells by hypoxic preconditioning.

BackgroundBone marrow stromal cells (BMSCs) are attractive as a source of neural progenitors for ex vivo generation of neurons and glia. Limited numbers of this subpopulation, however, hinder translation into autologous cell-based therapy. Here, we demonstrate rapid and efficient conditioning with hypoxia to enrich for these neural progenitor cells prior to further expansion in neurosphere culture.MethodAdherent cultures of BMSCs (rat/human) were subjected to 1 % oxygen for 24 h and then subcultured as neurospheres with epidermal growth factor (EGF) and basic fibroblast growth factor supplementation. Neurospheres and cell progeny were monitored immunocytochemically for marker expression. To generate Schwann cell-like cells, neurospheres were plated out and exposed to gliogenic medium. The resulting cells were co-cultured with purified dorsal root ganglia (rat) neurons and then tested for commitment to the Schwann cell fate. Fate-committed Schwann cells were subjected to in vitro myelination assay.ResultsTransient hypoxic treatment increased the size and number of neurospheres generated from both rat and human BMSCs. This effect was EGF-dependent and attenuated with the EGF receptor inhibitor erlotinib. Hypoxia did not affect the capacity of neurospheres to generate neuron- or glia-like precursors. Human Schwann cell-like cells generated from hypoxia-treated BMSCs demonstrated expression of S100β /p75 and capacity for myelination in vitro.ConclusionEnhancing the yield of neural progenitor cells with hypoxic preconditioning of BMSCs in vitro but without inherent risks of genetic manipulation provides a platform for upscaling production of neural cell derivatives for clinical application in cell-based therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0409-x) contains supplementary material, which is available to authorized users.

Human Bone Marrow Stromal Cells: A Reliable, Challenging Tool for In Vitro Osteogenesis and Bone Tissue Engineering Approaches.

Adult human bone marrow stromal cells (hBMSC) are important for many scientific purposes because of their multipotency, availability, and relatively easy handling. They are frequently used to study osteogenesis in vitro. Most commonly, hBMSC are isolated from bone marrow aspirates collected in clinical routine and cultured under the “aspect plastic adherence” without any further selection. Owing to the random donor population, they show a broad heterogeneity. Here, the osteogenic differentiation potential of 531 hBMSC was analyzed. The data were supplied to correlation analysis involving donor age, gender, and body mass index. hBMSC preparations were characterized as follows: (a) how many passages the osteogenic characteristics are stable in and (b) the influence of supplements and culture duration on osteogenic parameters (tissue nonspecific alkaline phosphatase (TNAP), octamer binding transcription factor 4, core-binding factor alpha-1, parathyroid hormone receptor, bone gla protein, and peroxisome proliferator-activated protein γ). The results show that no strong prediction could be made from donor data to the osteogenic differentiation potential; only the ratio of induced TNAP to endogenous TNAP could be a reliable criterion. The results give evidence that hBMSC cultures are stable until passage 7 without substantial loss of differentiation potential and that established differentiation protocols lead to osteoblast-like cells but not to fully authentic osteoblasts.

Autologous bone marrow stromal cell transplantation as a treatment for acute radiation enteritis induced by a moderate dose of radiation in dogs

Radiation enteritis is one of the most common complications of cancer radiotherapy, and the development of new and effective measures for its prevention and treatment is of great importance. Adult bone marrow stromal stem cells (ABMSCs) are capable of self-renewal and exhibit low immunogenicity. In this study, we investigated ABMSC transplantation as a treatment for acute radiation enteritis. We developed a dog model of acute radiation enteritis using abdominal intensity-modulated radiation therapy in a single X-ray dose of 14 Gy. ABMSCs were cultured in vitro, identified via immunofluorescence and flow cytometry, and double labeled with CM-Dil and superparamagnetic iron oxide (SPIO) before transplantation, which took place 48 hours after abdominal irradiation in a single fraction. The dog model of acute radiation enteritis was transplanted with cultured ABMSCs labeled with CM-Dil and SPIO into the mesenteric artery through the femoral artery. Compared with untreated control groups, dogs treated with ABMSCs exhibited substantially longer survival time and improved relief of clinical symptoms. ABMSC transplantation induced the regeneration of the intestinal epithelium and the recovery of intestinal function. Furthermore, ABMSC transplantation resulted in elevated serum levels of the anti-inflammatory cytokine interleukin-11 (IL10) and intestinal radioprotective factors, such as keratinocyte growth factor, basic fibroblast growth factor-2, and platelet-derived growth factor-B while reducing the serum level of the inflammatory cytokine IL17. ABMSCs induced the regeneration of the intestinal epithelium and regulated the secretion of serum cytokines and the expression of radioprotective proteins and thus could be beneficial in the development of novel and effective mitigators of and protectors against acute radiation enteritis.

Distribution and Viability of Fetal and Adult Human Bone Marrow Stromal Cells in a Biaxial Rotating Vessel Bioreactor after Seeding on Polymeric 3D Additive Manufactured Scaffolds.

One of the conventional approaches in tissue engineering is the use of scaffolds in combination with cells to obtain mechanically stable tissue constructs in vitro prior to implantation. Additive manufacturing by fused deposition modeling is a widely used technique to produce porous scaffolds with defined pore network, geometry, and therewith defined mechanical properties. Bone marrow-derived mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering-based cell therapies due to their multipotent character. One of the hurdles to overcome when combining additive manufactured scaffolds with MSCs is the resulting heterogeneous cell distribution and limited cell proliferation capacity. In this study, we show that the use of a biaxial rotating bioreactor, after static culture of human fetal MSCs (hfMSCs) seeded on synthetic polymeric scaffolds, improved the homogeneity of cell and extracellular matrix distribution and increased the total cell number. Furthermore, we show that the relative mRNA expression levels of indicators for stemness and differentiation are not significantly changed upon this bioreactor culture, whereas static culture shows variations of several indicators for stemness and differentiation. The biaxial rotating bioreactor presented here offers a homogeneous distribution of hfMSCs, enabling studies on MSCs fate in additive manufactured scaffolds without inducing undesired differentiation.

Onset of heterogeneity in culture-expanded bone marrow stromal cells

Inconsistencies among in vitro and in vivo experiments using adult mesenchymal stem cells (MSCs) confound development of therapeutic, regenerative medicine applications, and in vitro expansion is typically required to achieve sufficient cell numbers for basic research or clinical trials. Though heterogeneity in both morphology and differentiation capacity of culture-expanded cells is noted, sources and consequences are not well understood. Here, we endeavored to observe the onset of population heterogeneity by conducting long-term continuous in vitro observation of human adult bone marrow stromal cell (BMSC) populations, a subset of which has been shown to be stem cells (also known as bone marrow-derived MSCs). Semi-automated identification and tracking of cell division and migration enabled construction of cell lineage maps that incorporated cell morphology. We found that all BMSCs steadily grew larger over time; this growth was interrupted only when a cell divided, producing two equally sized, morphologically similar daughter cells. However, a finite probability existed that one or both of these daughters then continued to increase in size without dividing, apparently exiting the cell cycle. Thus, larger BMSCs are those cells that have exited the normal cell cycle. These results hold important implications for MSC in vitro culture expansion and biophysical sorting strategies.

Understanding the role of the microenvironment during definitive hemopoietic development

Hemopoietic stem cells (HSCs) are sustained in a specific microenvironment known as the stem cell niche. Recent studies in adult bone marrow have identified osteoblasts and endothelial cells as two important supportive cell types within the niche and demonstrated that interactions between HSCs and cellular and extracellular components within the endosteal and perivascular regions are critical for HSC regulation. However, the understanding of the role of the microenvironment in definitive HSC establishment, expansion, and maintenance during embryonic development is extremely limited. This review focuses on what is known about the components of each HSC microenvironment at various developmental stages and their known functional roles.

Isolation, culture and evaluation of multilineage-differentiating stress-enduring (Muse) cells

Multilineage-differentiating stress-enduring (Muse) cells are distinct stem cells in mesenchymal cell populations with the capacity to self-renew, to differentiate into cells representative of all three germ layers from a single cell, and to repair damaged tissues by spontaneous differentiation into tissue-specific cells without forming teratomas. We describe step-by-step procedures for isolating and evaluating these cells. Muse cells are also a practical cell source for human induced pluripotent stem (iPS) cells with markedly high generation efficiency. They can be collected as cells that are double positive for stage-specific embryonic antigen-3 (SSEA-3) and CD105 from commercially available mesenchymal cells, such as adult human bone marrow stromal cells and dermal fibroblasts, or from fresh adult human bone marrow samples. Under both spontaneous and induced differentiation conditions, they show triploblastic differentiation. It takes 4-6 h to collect and 2 weeks to confirm the differentiation and self-renewal capacity of Muse cells.

Polarized neural stem cells derived from adult bone marrow stromal cells develop a rosette-like structure

Bone marrow stromal cells (BMSCs) were reported to form floating aggregation of cells with expression of nestin, a marker for neural stem cells (NSCs). The purpose of this investigation is to evaluate the morphology and the molecular markers expressed by NSCs derived from these neurospheres. The BMSCs were isolated from Sprague Dawley rats and evaluated for osteogenesis, lipogenesis, and expression of fibronectin, CD90, CD106, CD31, and Oct4. The BMSCs were cultured with Dulbecco's modified Eagle's medium (DMEM)/F12 containing 15% fetal bovine serum, then with DMEM/F12 containing 2% B27, basic fibroblast growth factor, and epidermal growth factor. The cell aggregates or spheres were stained with acridine orange, which showed that the neurospheres comprised aggregated cells at either premitotic/postsynthetic (PS), postmitotic/presynthetic (PM) phases of cell cycle, or a mixture of both. The NSCs harvested from the neurospheres were polar with eccentric nucleus, and at either a PS or a PM cell cycle phases, some cells at the latter phase tended to form rosette-like structures. The cells were immunostained for molecular markers such as nestin, neurofilament 68 (NF68), NF160, and NF200 and glial fibrillary acidic protein (GFAP). Myelin basic protein (MBP), the pluripotency (Oct4, Nanog, and SOX2), and the differentiation genes (NeuroD1, Tubb4, and Musashi I) were also evaluated using reverse transcription polymerase chain reaction (RT-PCR). Nestin, NF68, NF160, NF200, GFAP, O4, and N-cadherin were expressed in the NSCs. The percentage of immunoreactive cells to nestin was significantly higher than that of the other neuronal markers. MBP was not expressed in BMSCs, neurospheres, and NSCs. The neurospheres were immunoreactive to GFAP. RT-PCR showed the expression of NeuroD1 and Musashi I. The pluripotency gene (SOX2) was expressed in NSCs. Oct4 and Nanog were expressed in BMSCs, while Oct4 and SOX2 were expressed in the neurosphere. This indicates that a pluripotency regularity network existed during the transdifferentiation of BMSCs into NSCs. Image processing of the neurospheres showed that the cells tended to form radial patterns. The conclusion of this study is that the NSCs generated from the BMSC-derived neurospheres have the morphology and the characteristics of neuroepithelial cells with tendency to forming rosette-like structures.

Injection Time-Dependent Effect of Adult Human Bone Marrow Stromal Cell Transplantation in a Rat Model of Severe Traumatic Brain Injury

The object of this study is to evaluate the effects of injecting adult human bone marrow stromal cells (hBMSCs) into rats with severe traumatic brain injury in acute phase and to determine more optimal injection timing between day 1 and day 2 postinjury. The lateral fluid percussion injury model was used. Adult hBMSCs were transplanted into hemisphere to injury sites in the corpus callosum ipsilateral on day 1 (n = 12) or day 7 (n = 8) after injury. A control group (n = 7) underwent only a sham operation without stem cell transplantation. Rats in all groups were analyzed by magnetic resonance spectroscopy (MRS), and by using behavioral, rotarod, and Barnes maze tests on day 1, 7, 14, and 42. Another nine randomly designated rats were sacrificed for immunohistochemical staining. Behavioral test scores increased significantly at all time-points after TBI in the day 7-injected group, compared to the others (p=0.008). GFAP staining was lower on day 42 in day 7-injected rats than in those injected on day 1. But no significant inter- or intra-group differences were observed for other tests. The injection of hBMSCs was found to have limited therapeutic potential with respect to neuroprotection after traumatic brain injury. However, because injection on day 7 after TBI produced greater functional improvements in neurobehavioral tests and more effectively suppressed astroglial activation than an injection on post-injury day 1, we cautiously recommend the injection time of day 7 post injury in hBMSCs transplantation in severe TBI, rather than day 1 post injury but further studies on developing hBMSC-based new therapeutic approaches should be warranted for improving neuroprotection in severe TBI.

Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells

Inflammatory cytokines present in the milieu of the fracture site are important modulators of bone healing. Here we investigated the effects of interleukin-1β (IL-1β) on the main events of endochondral bone formation by human bone marrow mesenchymal stromal cells (BM-MSC), namely cell proliferation, differentiation and maturation/remodelling of the resulting hypertrophic cartilage. Low doses of IL-1β (50 pg/mL) enhanced colony-forming units-fibroblastic (CFU-f) and -osteoblastic (CFU-o) number (up to 1.5-fold) and size (1.2-fold) in the absence of further supplements and glycosaminoglycan accumulation (1.4-fold) upon BM-MSC chondrogenic induction. In osteogenically cultured BM-MSC, IL-1β enhanced calcium deposition (62.2-fold) and BMP-2 mRNA expression by differential activation of NF-κB and ERK signalling. IL-1β-treatment of BM-MSC generated cartilage resulted in higher production of MMP-13 (14.0-fold) in vitro, mirrored by an increased accumulation of the cryptic cleaved fragment of aggrecan, and more efficient cartilage remodelling/resorption after 5 weeks in vivo (i.e., more TRAP positive cells and bone marrow, less cartilaginous areas), resulting in the formation of mature bone and bone marrow after 12 weeks. In conclusion, IL-1β finely modulates early and late events of the endochondral bone formation by BM-MSC. Controlling the inflammatory environment could enhance the success of therapeutic approaches for the treatment of fractures by resident MSC and as well as improve the engineering of implantable tissues.