Mesenchymal stem cells (MSC) are adult multipotent cells found in bone marrow, adipose tissue, and other adult tissues. MSC have been shown to improve regeneration of injured tissues in vivo, but the mechanisms remain unclear. Typically, MSC are cultured under ambient, or normoxic, conditions (21% oxygen). However, the physiological niches for MSC in the bone marrow and other sites have much lower oxygen tension. When used as a therapeutic tool to repair tissue injuries, MSC cultured in standard conditions must adapt from 21% oxygen in culture to less than 1% oxygen in the ischemic tissue. We therefore examined the effects of preculturing human bone marrow-derived MSC in hypoxic conditions (1%–3% oxygen) to elucidate the best conditions that enhance their tissue regenerative potential. We demonstrated that MSC cultured in hypoxia activate the Akt signaling pathway while maintaining their viability and cell cycle rates. We also showed that MSC cultured in hypoxia induced expression of cMet, the major receptor for hepatocyte growth factor (HGF), and enhanced cMet signaling. MSC cultured in hypoxic conditions increased their migration rates. Since migration and HGF responsiveness are thought to be key mediators of MSC recruitment and/or activation in vivo, we next examined the tissue regenerative potential of MSC cultured under hypoxic conditions, using a murine hind limb ischemia model. We showed that local expression of HGF is increased in ischemic muscle in this model. Intra-arterial injection of MSC cultured in either normoxic or hypoxic conditions 24 hours after surgical induction of hind limb ischemia enhanced revascularization compared with saline controls. However, restoration of blood flow was observed significantly earlier in mice that had been injected with hypoxic preconditioned MSC. Collectively, these data suggest that preculturing MSC under hypoxic conditions prior to transplantation improves their tissue regenerative potential.
Immune-deficient mice; Human stem cells; Mesenchymal stem cells; Hypoxia; Transplantation; Tissue repair
Mesenchymal stem cells (MSCs) derived from bone marrow (BM), adipose tissue (AT), umbilical cord blood (CB), and umbilical cord tissue (CT) are increasingly being used to treat equine inflammatory and degenerative lesions. MSCs modulate the immune system in part through mediator secretion. Animal species and MSC tissue of origin are both important determinants of MSC function. In spite of widespread clinical use, how equine MSCs function to heal tissues is fully unknown. In this study, MSCs derived from BM, AT, CB, and CT were compared for their ability to inhibit lymphocyte proliferation and secrete mediators in response to activation. Five MSC lines from each tissue were isolated. Lymphocyte proliferation was assessed in a mixed leukocyte reaction, and mediator secretion was determined by ELISA. Regardless of tissue of origin, quiescent MSCs did not alter lymphocyte proliferation or secrete mediators, except for transforming growth factor-β (TGF-β1). When stimulated, MSCs of all tissue types decreased lymphocyte proliferation, increased prostaglandin (PGE2) and interleukin-6 (IL-6) secretion, and decreased production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). BM-MSCs and CB-MSCs also produced nitric oxide (NO), while AT-MSCs and CT-MSCs did not. Equine MSCs did not produce indoleamine 2,3-dioxygenase (IDO). These data suggest that activated equine MSCs derived from BM, AT, CT, and CB secrete high concentration of mediators and are similar to MSCs from rodents and humans in their immunomodulatory profiles. These findings have implication for the treatment of inflammatory lesions dominated by activated lymphocytes and TNF-α and IFN-γ in vivo.
Equine; Mesenchymal stem cells; Immunomodulation; Lymphocytes; Bone marrow; Umbilical cord blood; Adipose and umbilical cord tissue
Mesenchymal stem cells (MSCs) are multipotent cells that have the capacity to develop into different mature mesenchymal cell types. They were originally isolated from bone marrow, but MSC-like cells have also been isolated from other tissues. The common feature of all of these tissues is that they all house blood vessels. It is, thus, possible that MSCs are associated with perivascular locations. The objective of this work was to test the hypothesis that MSCs are associated with blood vessels by verifying if MSC frequency positively correlates with blood vessel density. To this end, samples from highly and poorly vascularized adipose tissue sites of two equine donors were collected and processed for histology and cell isolation. MSC frequency in these samples was estimated by means of CFU-F assays, which were performed under MSC conditions. Culture-adherent cells from equine adipose tissue and bone marrow were culture expanded, tested for differentiation into mesenchymal cell types in vitro, and implanted in vivo in porous ceramic vehicles to assess their osteogenic capacity, using human MSCs and brain pericytes as controls. The differentiation assays showed a difference between adipose tissue–derived cells as compared to equine bone marrow MSCs. While differences in CFU-F frequencies between both donors were evident, the CFU-F numbers correlated directly with blood vessel densities (r2 = 0.86). We consider these preliminary data as further evidence linking MSCs to blood vessels.
Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy and can be isolated from various sources of human adult tissues such as bone marrow (BM-MSC) and adipose tissue. However, cells from these tissues must be obtained through invasive procedures. We, therefore, characterized MSCs isolated from fresh placenta (Pl-MSC) and fetal membrane (Mb-MSC) through morphological and fluorescent-activated cell sorting (FACS). MSC frequency is higher in membrane than placenta (2.14% ± 0.65 versus 15.67% ± 0.29%). Pl/Mb-MSCs in vitro expansion potential was significantly higher than BM-MSCs. We demonstrated that one of the MSC-specific marker is sufficient for MSC isolation and that culture in specific media is the optimal way for selecting very homogenous MSC population. These MSCs could be differentiated into mesodermal cells expressing cell markers and cytologic staining consistent with mature osteoblasts and adipocytes. Transcriptomic analysis and cytokine arrays demonstrated broad similarity between placenta- and membrane-derived MSCs and only discrete differences with BM-MSCs with enrichment of networks involved in bone differentiation. Pl/Mb-MSCs displayed higher osteogenic differentiation potential than BM-MSC when their response to osteoactivin was evaluated. Fetal-tissue-derived mesenchymal cells may, therefore, be considered as a major source of MSCs to reach clinical scale banking in particular for bone regeneration.
Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.
After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1α expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.
Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.
Mesenchymal stromal cells (MSCs) reside in many organs including lung, as shown by their isolation from fetal lung tissues, bronchial stromal compartment, bronchial-alveolar lavage and transplanted lung tissues. It is still controversial whether lung MSCs can undergo mesenchymal-to-epithelial-transition (MET) and possess immune regulatory properties. To this aim, we isolated, expanded and characterized MSCs from normal adult human lung (lung-hMSCs) and compared with human bone marrow-derived MSCs (BM-hMSCs). Our results show that lung-MSCs reside at the perivascular level and do not significantly differ from BM-hMSCs in terms of immunophenotype, stemness gene profile, mesodermal differentiation potential and modulation of T, B and NK cells. However, lung-hMSCs express higher basal level of the stemness-related marker nestin and show, following in vitro treatment with retinoic acid, higher epithelial cell polarization, which is anyway partial when compared to a control epithelial bronchial cell line. Although these results question the real capability of acquiring epithelial functions by MSCs and the feasibility of MSC-based therapeutic approaches to regenerate damaged lung tissues, the characterization of this lung-hMSC population may be useful to study the involvement of stromal cell compartment in lung diseases in which MET plays a role, such as in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.
Bone marrow-derived mesenchymal stem cells (BM-MSCs) and adipose tissue-derived mesenchymal stem cells (AT-MSCs) are potential cellular sources of therapeutic stem cells. MSCs are a multipotent population of cells capable of differentiating into a number of mesodermal lineages. Treatment using MSCs appears to be a helpful approach for structural restoration in regenerative medicine. Correct identification of these cells is necessary, but there is inadequate information on the MSC profile of cell surface markers and mRNA expression in dogs. In this study, we performed molecular characterization of canine BM-MSCs and AT-MSCs using immunological and mRNA expression analysis.
Samples were confirmed to be multipotent based on their osteogenic and adipogenic differentiation. And these cells were checked as stem cell, hematopoietic and embryonic stem cell (ESC) markers by flow cytometry. BM- and AT-MSCs showed high expression of CD29 and CD44, moderate expression of CD90, and were negative for CD34, CD45, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. SSEA-1 was expressed at very low levels in AT-MSCs. Quantitative real-time PCR (qRT-PCR) revealed expression of Oct3/4, Sox2, and Nanog in BM- and AT-MSCs. There was no significant difference in expression of Oct3/4 and Sox2 between BM-MSCs and AT-MSCs. However, Nanog expression was 2.5-fold higher in AT-MSCs than in BM-MSCs. Using immunocytochemical analysis, Oct3/4 and Sox2 proteins were observed in BM- and AT-MSCs.
Our results provide fundamental information to enable for more reproducible and reliable quality control in the identification of canine BM-MSCs and AT-MSCs by protein and mRNA expression analysis.
Canine; Mesenchymal stem cell; Cell surface markers; Embryonic stem cell markers
Mesenchymal stem cells (MSC) derived from bone marrow stem cells (BMSC) and adipose tissue stem cells (ASC) of humans and rhesus macaques were evaluated for their cell cycle properties during protracted culture in vitro. Human ASCs (hASC) and rhesus BMSCs (rBMSC) underwent significantly more total population doublings than human BMSCs (hBMSC) and rhesus ASCs (rASC). The cell cycle profile of all MSCs was altered as cultures aged. hMSCs underwent an increase in the frequency of cells in the S phase at P20 and P30. However, rhesus MSCs from both sources developed a distinct polyploid population of cells at P20, which progressed to aneuploidy by P30. Karyotype analysis of MSCs revealed the development of tetraploid or aneuploid karyotypes in the rhesus cells at P20 or P30. Analysis of the transcriptome of the MSCs from early and late passages revealed significant alterations in the patterns of gene expression (8.8% of the genes were differentially expressed in hBMSCs versus hASCs, and 5.5% in rBMSCs versus rASCs). Gene expression changes were much less evident within the same cell type as aging occurred (0.7% in hMSCs and 0.9% in rMSC). Gene ontology analysis showed that functions involved in protein catabolism and regulation of pol II transcription were overrepresented in rASCs, whereas the regulation of IκB/nuclear factor-κB cascade were overrepresented in hBMSCs. Functional analysis of genes that were differentially expressed in rASCs and hBMSCs revealed that pathways involved in cell cycle, cell cycle checkpoints, protein-ubiquitination, and apoptosis were altered.
There has been a recent increase in our understanding in the isolation, culture, and differentiation of mesenchymal stem cells (MSCs). Concomitantly, the availability of MSCs has increased, with cells now commercially available, including human MSCs from adipose tissue and bone marrow. Despite an increased understanding of MSC biology and an increase in their availability, standardization of techniques for adipogenic differentiation of MSCs is lacking. The following review will explore the variability in adipogenic differentiation in vitro, specifically in 3T3-L1 and primary MSCs derived from both adipose tissue and bone marrow. A review of alternative methods of adipogenic induction is also presented, including the use of specific peroxisome proliferator-activated receptor-gamma agonists as well as bone morphogenetic proteins. Finally, we define a standard, commonly used adipogenic differentiation medium in the hopes that this will be adopted for the future standardization of laboratory techniques—however, we also highlight the essentially arbitrary nature of this decision. With the current, rapid pace of electronic publications, it becomes imperative for standardization of such basic techniques so that interlaboratory results may be easily compared and interpreted.
Alternative sources of mesenchymal stem cells (MSCs) for replacing bone marrow (BM) have been extensively investigated in the field of bone tissue engineering. The purpose of this study was to compare the osteogenic potential of canine MSCs derived from adipose tissue (AT), BM, umbilical cord blood (UCB), and Wharton's jelly (WJ) using in vitro culture techniques and in vivo orthotopic implantation assays. After canine MSCs were isolated from various tissues, the proliferation and osteogenic potential along with vascular endothelial growth factor (VEGF) production were measured and compared in vitro. For the in vivo assay, MSCs derived from each type of tissue were mixed with β-tricalcium phosphate and implanted into segmental bone defects in dogs. Among the different types of MSCs, AT-MSCs had a higher proliferation potential and BM-MSCs produced the most VEGF. AT-MSCs and UCB-MSCs showed greater in vitro osteogenic potential compared to the other cells. Radiographic and histological analyses showed that all tested MSCs had similar osteogenic capacities, and the level of new bone formation was much higher with implants containing MSCs than cell-free implants. These results indicate that AT-MSCs, UCB-MSCs, and WJ-MSCs can potentially be used in place of BM-MSCs for clinical bone engineering procedures.
cell source; dogs; mesenchymal stem cells; osteogenesis
Mesenchymal stem cells (MSCs) are the most popular among the adult stem cells in tissue engineering and regenerative medicine. Since their discovery and functional characterization in the late sixties and early seventies, MSCs or MSC-like cells have been obtained from various mesodermal and non-mesodermal tissues, although majority of the therapeutic applications involved bone marrow derived MSCs. Based on its mesenchymal origin, it was predicted earlier that MSCs only can differentiate into mesengenic lineages like bone, cartilage, fat or muscle. However, varied isolation and cell culturing methods identified subsets of MSCs in the bone marrow which not only differentiated into mesenchymal lineages, but also into ectodermal and endodermal derivatives. Although, true pluripotent status is yet to be established, MSCs have been successfully used in bone and cartilage regeneration in osteoporotic fracture and arthritis respectively and in the repair of cardiac tissue following myocardial infarction. Immunosuppressive properties of MSCs extend utility of MSCs to reduce complications of graft versus host disease and rheumatoid arthritis. Homing of MSCs to sites of tissue injury, including tumor, is well established. In addition to their ability in tissue regeneration, MSCs can be genetically engineered ex vivo for delivery of therapeutic molecule(s) to the sites of injury or tumorigenesis as cell therapy vehicles. MSCs tend to lose surface receptors for trafficking and have been reported to develop sarcoma in long-term culture. In this article, we reviewed the current status of MSCs with special emphasis to therapeutic application in bone-related diseases.
Human induced pluripotent stem cells (iPSCs) possess remarkable self-renewal capacity and the potential to differentiate into novel cell types, such as mesenchymal stem cells (MSCs). iPSC-MSCs have been shown to enhance tissue regeneration and attenuate tissue ischaemia; however, their contribution to the immune regulation of Th2-skewed allergic rhinitis (AR) and asthma remains unclear.
This study compared the immunomodulatory effects of iPSC-MSCs and bone marrow-derived MSCs (BM-MSCs) on lymphocyte proliferation, T-cell phenotypes and cytokine production in peripheral blood mononuclear cells (PBMCs) in patients with AR, and investigated the possible molecular mechanisms underlying the immunomodulatory properties of iPSC-MSCs.
In co-cultures of PBMCs with iPSC-MSCs or BM-MSCs, lymphocyte proliferation was evaluated using 3H-thymidine (3H-TdR) uptake, carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE) assays; the regulatory T-cell (Treg) phenotype was determined by flow cytometry, and cytokine levels were measured using an enzyme-linked immunosorbent assay. The immunomodulatory properties of both MSCs were further evaluated using NS398 and transwell experiments.
Similar to BM-MSCs, we determined that iPSC-MSCs significantly inhibit lymphocyte proliferation and promote Treg response in PBMCs (P < 0.05). Accordingly, the cytokine milieu (IFN-γ, IL-4, IL-5, IL-10 and IL-13) in the supernatants of PBMCs changed significantly (P < 0.05). The immunomodulatory properties of iPSC-MSCs and BM-MSCs were associated with prostaglandin E2 (PGE2) production and cell–cell contact.
These data demonstrate that iPSC-MSCs are capable of modulating T-cell phenotypes towards Th2 suppression through inducing Treg expansion, suggesting that iPSC-MSCs can be used as an alternative candidate to adult MSCs to treat allergic airway diseases.
allergic rhinitis; immunomodulation; induced pluripotent stem cells; mesenchymal stem cells; T cell
To ensure the survival of engineered bone after implantation, we combined human endothelial colony forming cells (ECFCs) and multipotent stromal cells (MSCs) as a proof of concept in a co-culture model to create in vitro prevascularized bone constructs. We hypothesized that a hypoxic stimulus will contribute to prevascularization of engineered bone. Bone marrow-derived MSCs and ECFCs from human adult peripheral blood were allowed to form co-culture pellets containing ECFCs and MSCs (1:4) or MSCs only in controls. After culture under normoxia or hypoxia (5%), pellets were harvested and processed for immunohistochemistry of CD31, α-smooth muscle actin, and osteocalcin. Expression of vascular endothelial growth factor and SDF-1α was analyzed by PCR to elucidate their involvement in hypoxic stimulation of prevascularization. The normoxic condition in co-cultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells. These structures failed to form in hypoxic conditions, thereby rejecting the hypothesis that hypoxia stimulates prevasculogenesis in three-dimensional engineered bone constructs. Further, the formation of prevascular structures was paralleled by increased SDF-1α expression. It is suggested that actual oxygen levels were below 5% in the hypoxic co-cultures, which prevented prevascular structure formation. In conclusion, our normoxic co-culture model containing cells from clinically relevant sources sustained simultaneous endothelial, smooth muscle, and osteogenic differentiation.
In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions.
Morphologic studies, differentiation and transcriptional profiling experiments were performed on MSC cultured in normoxia (21% O2) versus hypoxia (5% O2) for up to passage 2. Cells at passage 0 and at passage 2 were compared, and those at passage 0 in hypoxia generated fewer and smaller colonies than in normoxia. In parallel, MSC displayed (>4 fold) inhibition of genes involved in DNA metabolism, cell cycle progression and chromosome cohesion whereas transcripts involved in adhesion and metabolism (CD93, ESAM, VWF, PLVAP, ANGPT2, LEP, TCF1) were stimulated. Compared to normoxic cells, hypoxic cells were morphologically undifferentiated and contained less mitochondrias. After this lag phase, cells at passage 2 in hypoxia outgrew the cells cultured in normoxia and displayed an enhanced expression of genes (4-60 fold) involved in extracellular matrix assembly (SMOC2), neural and muscle development (NOG, GPR56, SNTG2, LAMA) and epithelial development (DMKN). This group described herein for the first time was assigned by the Gene Ontology program to "plasticity".
The duration of hypoxemia is a critical parameter in the differentiation capacity of MSC. Even in growth promoting conditions, hypoxia enhanced a genetic program that maintained the cells undifferentiated and multipotent. This condition may better reflect the in vivo gene signature of MSC, with potential implications in regenerative medicine.
Mesenchymal stem cells/marrow stromal cells (MSC) are adult multipotent cells that can augment tissue repair. We previously demonstrated that culturing MSC in hypoxic conditions causes upregulation of the hepatocyte growth factor (HGF) receptor c-Met, allowing them to respond more robustly to HGF. MSC preconditioned in hypoxic environments contributed to restoration of blood flow after an ischemic injury more rapidly than MSC cultured in normoxic conditions. We now investigated the specific role of HGF/c-Met signaling in MSC function. An shRNA-mediated knockdown (KD) of c-Met in MSC did not alter their phenotypic profile, proliferation, or viability in vitro. However, we determined that while HGF/c-Met signaling does not play a role in the adipogenic differentiation of the cells, the disruption of this signaling pathway inhibited the ability of MSC to differentiate into the osteogenic and chondrogenic lineages. We next assessed the impact of c-Met KD on human MSC function in a xenogeneic hindlimb ischemia injury model. A 70% KD of c-Met in MSC resulted in a significant decrease in their capacity to regenerate blood flow to the ischemic limb, as compared to the MSC transduced with control shRNA. MSC with only a 60% KD of c-Met exhibited an intermediate capacity to restore blood flow, suggesting that MSC function is sensitive to the dosage of c-Met signaling. The current study highlights the significance of HGF/c-Met signaling in the capacity of MSC to restore blood flow after an ischemic injury and in their ability to differentiate into the osteogenic and chondrogenic lineages.
The ability of stem/progenitor cells to migrate and engraft into host tissues is key to their potential use in gene and cell therapy. Among the cells of interest are the adherent cells from bone marrow, referred to as mesenchymal stem cells or multipotent stromal cells (MSC). Since the bone marrow environment is hypoxic, with oxygen tensions ranging from 1% to 7%, we decided to test whether hypoxia can upregulate chemokine receptors and enhance the ability of human MSCs to engraft in vivo. Short-term exposure of MSCs to 1% oxygen increased expression of the chemokine receptors CX3CR1and CXCR4, both as mRNA and as protein. After 1-day exposure to low oxygen, MSCs increased in vitro migration in response to the fractalkine and SDF-1α in a dose dependent manner. Blocking antibodies for the chemokine receptors significantly decreased the migration. Xenotypic grafting into early chick embryos demonstrated cells from hypoxic cultures engrafted more efficiently than cells from normoxic cultures and generated a variety of cell types in host tissues. The results suggest that short-term culture of MSCs under hypoxic conditions may provide a general method of enhancing their engraftment in vivo into a variety of tissues.
Adipose tissue represents an accessible, abundant, and replenishable source of adult stem cells for potential applications in regenerative medicine. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) resemble bone marrow-derived mesenchymal stem cells (BM-MSCs) with respect to morphology, immune-phenotype and multiple differentiation capability. In the present study, we investigated the feasibility of AT-MSC-based liver gene delivery for the treatment of alpha 1-antitrypsin deficiency.
Mouse AT-MSCs were tranduced by rAAV vectors and transplanted into mouse liver
We showed that AT-MSCs can be transduced by recombinant adeno-associated viral vector serotype 1 (rAAV1-CB-hAAT). After transplanting to mouse liver, ex vivo transduced AT-MSCs expressed the transgene product, human alpha 1-antitrypsin (hAAT). Importantly, serum levels of hAAT were sustained and no anti-hAAT antibody was detected in any recipients.
These results demonstrated that AT-MSCs can be transduced by rAAV vectors, engrafted into recipient livers, contribute to liver regeneration, and serve as a platform for transgene expression without eliciting an immune response. AT-MSC-based gene therapy presents a novel approach for the treatment of liver diseases, such as AAT deficiency.
Alpha 1 antitrypsin deficiency; Adeno-associated virus (AAV); Liver gene therapy; Liver regeneration
Present in numerous tissues, mesenchymal stem cells/multipotent stromal cells (MSCs) can differentiate into different cell types from a mesoderm origin. Their potential has been extended to pluripotency, by their possibility of differentiating into tissues and cells of nonmesodermic origin. Through the release of cytokines, growth factors and biologically active molecules, MSCs exert important paracrine effects during tissue repair and inflammation. Moreover, MSCs have immunosuppressive properties related to non-HLA restricted immunosuppressive capacities. All these features lead to an increasing range of possible applications of MSCs, from treating immunological diseases to tissue and organ repair, that should be tested in phase I and II clinical trials. The most widely used MSCs are cultured from bone marrow or adipose tissue. For clinical trial implementation, BM MSCs and ADSCs should be produced according to Good Manufacturing Practices. Safety remains the major concern and must be ensured during culture and validated with relevant controls. We describe some applications of MSCs in clinical trials.
Adipose-derived stem cells (ADSCs) are mesenchymal stem cells (MSCs) that can be extracted from adipose tissue and obtained by a less invasive method and in larger quantities compared with bone marrow–derived MSCs. The objective of this study was to harvest ADSCs from piglets and to explore their neuronal differentiation potential.
Adipose tissue from piglet facial or abdominal fat was digested with collagenase type XI, followed by filter and centrifugation; the isolated adipose stromal cells were cultured in dishes. MSC markers were measured by flow cytometry; 2 to 5 passage cells were used for in vitro differentiation. Adipogenic, chondrogenic, osteogenic, and neuronal differentiation was induced by incubation of the ADSCs with different induction media.
ADSCs were easily expanded to beyond 15 passages, maintaining the undifferentiated state and exhibiting MSC characteristics and markers CD29, CD44, and CD90. ADSCs differentiated into other mesodermal cells including adipocytes, chondrocytes, and osteocytes. These cells were induced to differentiate into neuron-like cells as evidenced by neuronal morphology and the presence of neuronal markers including microtubule-associated protein 2, neuronal nuclear antigen, and β-tubulin III.
ADSCs can be readily obtained from a small amount fat tissue and expanded in culture. Adipose tissue may be an alternative source of stem cell therapy for nervous system injury.
Mesenchymal stem cell; Adipose tissue; Neuronal differentiation
Introduction. Bone-marrow-derived mesenchymal stem cells (MSCs) have been studied for treatment of myocardial infarction (MI). Since MSCs from older donors show quantitative and qualitative senescent changes, we hypothesized that a better outcome may be achieved if aged recipients are given MSCs obtained from young donors, rather than using their own autologous MSCs.
Methods. In vitro studies compared properties of young and old MSCs. Aged rats randomized into 3 groups underwent coronary artery ligations and were then injected with either old (O) or young (Y) MSCs, or ligation alone. Echocardiography evaluated ejection fractions (EF). At 16 weeks, scar deposition was analyzed. Results. Old MSCs exhibited decreased cell viability, proliferation, and differentiation potentials. EF significantly improved early in both cell therapy groups (P < .05). However, at later stages of the study, group Y showed significantly better function which correlated with decreased scar deposition.
Conclusions. The significant difference between young and old cells indicates the possible advantage for allotransplanting MSCs from young donors to elderly patients with MI.
Human stromal (mesenchymal) stem cells (hMSCs) are multipotent stem cells with ability to differentiate into mesoderm-type cells e.g. osteoblasts and adipocytes and thus they are being introduced into clinical trials for tissue regeneration. Traditionally, hMSCs have been isolated from bone marrow, but the number of cells obtained is limited. Here, we compared the MSC-like cell populations, obtained from alternative sources for MSC: adipose tissue and skin, with the standard phenotype of human bone marrow MSC (BM-MSCs). MSC from human adipose tissue (human adipose stromal cells (hATSCs)) and human skin (human adult skin stromal cells, (hASSCs) and human new-born skin stromal cells (hNSSCs)) grew readily in culture and the growth rate was highest in hNSSCs and lowest in hATSCs. Compared with phenotype of hBM-MSC, all cell populations were CD34−, CD45−, CD14−, CD31−, HLA-DR−, CD13+, CD29+, CD44+, CD73+, CD90+,and CD105+. When exposed to in vitro differentiation, hATSCs, hASSCs and hNSSCs exhibited quantitative differences in their ability to differentiate into adipocytes and to osteoblastic cells. Using a microarray-based approach we have unveiled a common MSC molecular signature composed of 33 CD markers including known MSC markers and several novel markers e.g. CD165, CD276, and CD82. However, significant differences in the molecular phenotype between these different stromal cell populations were observed suggesting ontological and functional differences. In conclusion, MSC populations obtained from different tissues exhibit significant differences in their proliferation, differentiation and molecular phenotype, which should be taken into consideration when planning their use in clinical protocols.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-012-9365-8) contains supplementary material, which is available to authorized users.
Stromal cells; Mesenchymal stem cell; Adipose tissue; Bone marrow; Skin; DNA microarray
Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages.
Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis.
Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches.
The potential translation of mesenchymal stem cell (MSC) therapy into a multimodal protocol for traumatic brain injury requires evaluation of viability and cytokine production in a hyperosmolar environment. Optimization of MSC therapy requires delivery to the target area without significant loss of cellular function or viability. No model evaluating the potential efficacy of MSC therapy at varying osmolarities currently exists.
Rat MSCs were characterized with flow cytometric immunophenotyping. MSCs (passage 3) were placed in culture with multipotent adult progenitor cell media at varying osmolarities (250, 270, 290, 310, 330, 350 and 370 mOsm) potentially found with hypertonic saline infusion. After culture for 24 h, cellular viability was measured using flow cytometry (n = 6). Next, brain tissue supernatant was harvested from both normal rat brains and injured brains 6 h after cortical injury. Subsequently, MSCs were placed in culture with multipotent adult progenitor cell media ± 20% normal brain or injured brain supernatant (at the aforementioned osmolarities) and allowed to remain in culture for 24 h (n = 11). At this point, media supernatant cytokine levels were measured using a multiplex cytokine assay system.
MSCs showed no clinically significant difference in viability at 24 h. MSCs cultured with 20% injured brain supernatant showed an decrease in proinflammatory cytokine production (IL-1α and IL-1β) with increasing osmolarity. No difference in anti-inflammatory cytokine production (IL-4 and IL-10) was observed.
Progenitor cell therapy for traumatic brain injury may require survival and activity in a hyperosmolar environment. Culture of MSCs in such conditions shows no clinically significant effect on cell viability. In addition, MSC efficacy could potentially be enhanced via a decrease in proinflammatory cytokine production. Overall, a multimodal traumatic brain injury treatment protocol based upon MSC infusion and hypertonic saline therapy would not negatively affect progenitor cell efficacy and could be considered for multicenter clinical trials.
cytokines; inflammation; mesenchymal stem cells; osmolarity
Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.
Mesenchymal stem cells (MSCs) originally isolated from bone marrow, are fibroblast-looking cells that are now assumed to be present in the stromal component of many tissues. MSCs are characterized by a certain set of criteria including their growth culture characteristics, a combination of cell surface markers, and the ability to differentiate along multiple mesenchymal tissue lineages. We hypothesized that human vocal fold fibroblasts (hVFF) isolated from the lamina propria meet the criteria established to define MSCs and are functionally similar to MSCs derived from BM and adipose tissue.
In vitro study
HVFF were previously derived from human vocal fold tissues. MSCs were derived from adipose tissue (AT), and BM of healthy donors, based on their attachment to culture dishes and their morphology, and expanded in culture. Cells were analyzed for standard cell surface markers identified on BM-derived MSCs as well as the ability to differentiate into cells of mesenchymal lineage, i.e. fat, bone and cartilage. We investigated the immunophenotype of these cells before and after interferon-γ (INF- γ) stimulation.
HVFF displayed cell surface markers and multipotent differentiation capacity characteristic of MSCs. Furthermore, these cells exhibited similar patterns of expression of HLA and co-stimulatory molecules, after stimulation with INF- γ compared to MSCs derived from BM and AT.
Based on our findings hVFF derived from lamina propria have the same cell surface markers, immunophenotypic characteristics, and differentiation potential as BM- and AT-derived MSCs. We propose VF fibroblasts are MSCs resident in the vocal fold lamina propria.
mesenchymal stem cells; human vocal fold stem cells; immune modulation; vocal fold fibroblasts