Alternative Source Of Mesenchymal Stem Cells Research Articles

Isolation and characterization of multipotent mesenchymal stem cells in nasal polyps

Mesenchymal stem cells (MSCs) are multipotent progenitor cells in adult tissues. This study aimed to investigate nasal polyp (NP) tissues as a potential new source of multipotent MSCs that maintain their stemness and differentiation potential following multiple rounds of passaging. NP tissues were obtained from 10 patients during endoscopic sinus surgery. After isolating and culturing NP-derived MSCs (npMSCs), the expression levels of the surface markers CD34, CD44, CD45, CD73, CD90, CD105, CD106, CD146 and human leukocyte antigens-class II DR antigen (HLA-DR) were estimated by flow cytometry. NpMSCs were cultured in chondrogenic, osteogenic, adipogenic, or neurogenic differentiation medium. The differentiation potential of npMSCs was analyzed by Alcian blue, alizarin red S, oil red O, and immunocytochemical staining and reverse transcription-polymerase chain reaction. The clonogenic potential of npMSCs was measured using a colony-forming unit assay. Cell proliferation of npMSCs was measured using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay. Flow cytometry analysis revealed that npMSCs were negative for hematopoietic lineage markers (CD34, CD45, and HLA-DR) and positive for MSC markers (CD44, CD73, CD90, and CD105). The npMSCs differentiated into osteogenic, adipogenic, chondrogenic, and neurogenic lineages, respectively. Chondrogenically differentiated npMSCs were stained with Alcian blue, osteogenically differentiated npMSCs were stained with alizarin red S, and adipogenically differentiated npMSCs were stained with oil red O. Real-time polymerase chain reaction results showed that the differentiated npMSCs expressed the respective differentiation markers (Sox 9 and Col2A for chondrogenesis, Runx2 and osteocalcin for osteogenesis, fatty acid-binding protein 4 and peroxisome proliferator-activated receptor γ for adipogenesis, TuJ1, neurofilament light chain, and neurofilament heavy chain for neurogenesis). There were no significant differences in the clonogenic potential and proliferation rate between early and late passage npMSCs. These results show that npMSCs possess the characteristics of MSCs in terms of morphology, multipotent differentiation capacity, cell surface marker expression, and clonogenicity. Thus, npMSCs may represent an alternative source of MSCs.

Bone marrow mesenchymal stem cell aspirates from alternative sources Is the knee as good as the iliac crest?

The most common method to obtain human mesenchymal stem cells (MSCs) is bone marrow aspiration from the iliac crest, but MSCs have also been isolated from different bones. The main purpose of this study was to compare bone marrow MSCs aspirated from the metaphysis of the distal femur and the proximal tibia with those obtained from the iliac crest, and to determine whether these locations represent potential alternative sources of MSCs for research and clinical application. Bone marrow was aspirated from the iliac crest and the metaphysis of the distal femur and the proximal tibia during total knee arthroplasty in 20 patients. The aspirates were centrifuged by density gradient, then mononucleated cell (MNC) concentration in the different aspirates was determined using a Coulter counter. MSCs were isolated, cultivated and characterised by their immunophenotype and by their in vitro potential for differentiation into osteoblasts, chondroblasts and adipocytes in specific media. Expansion and cell viability were quantified using trypan blue staining and cell counting with a haemocytometer (Neubauer chamber). The three sources were compared in terms of MNC concentration, viability of the cultures and presence of MSC using the Wilcoxon test. MNC concentration was significantly higher in the iliac crest (10.05 Millions/ml) compared with the femur (0.67 Millions/ml) and tibia (1.7 Millions/ml). Culture success rates were 90%, 71% and 47% for MSCs from the iliac crest, femur and tibia, respectively. Flow cytometry analysis showed the presence of CD90+, CD105+, CD73+, VEGF+, CD71+, HLA-DR-, CD45-, CD34-, CD19-, and CD14- cells. The immunophenotype pattern of MSCs was similar for the three locations. Trilineage differentiation was achieved with all samples. MSCs can be found in bone marrow from the metaphysis of both the distal femur and the proximal tibia. The phenotype and differentiation potential of these cells are similar to those of bone marrow MSCs from the iliac crest. Bone marrow aspiration from these locations is a relatively easy and safe alternative to that from the iliac crest for obtaining MSCs. Further study is required to assess whether the concentrations of MSCs obtained from these sources are sufficient for one-step therapeutic purposes.

Human embryonic stem cell derived-mesenchymal stem cells: an alternative mesenchymal stem cell source for regenerative medicine therapy.

To enumerate and characterize mesenchymal stem cells (MSC) derived from human embryonic stem cells (hESC) for clinical application. hESC were differentiated into hESC-MSC and characterized by the expression of surface markers using flow cytometry. hESC-MSC were evaluated with respect to growth kinetics, colony-forming potential, as well as osteogenic and adipogenic differentiation capacity. Immunosuppressive effects were assessed using peripheral blood mononuclear cell (PBMC) proliferation and cytotoxicity assays. hESC-MSC showed similar morphology, and cell surface markers as adipose (AMSC) and bone marrow-derived MSC (BMSC). hESC-MSC exhibited a higher growth rate during early in vitro expansion and equivalent adipogenic and osteogenic differentiation and colony-forming potential as AMSC and BMSC. hESC-MSC demonstrated similar immunosuppressive effects as AMSC and BMSC. hESC-MSC were comparable to BMSC and AMSC and hence can be used as an alternative source of MSC for clinical applications.

The osteoblastogenesis potential of adipose mesenchymal stem cells in myeloma patients who had received intensive therapy.

Multiple myeloma (MM) is characterized by advanced osteolytic lesions resulting from the activation of osteoclasts (OCs) and inhibition of osteoblasts (OBs). OBs are derived from mesenchymal stem cells (MSCs) from the bone marrow (BM), however the pool and function of BMMSCs in MM patients (MM-BMMSCs) are reduced by myeloma cells (MCs) and cytokines secreted from MCs and related anti-MM treatment. Such reduction in MM-BMMSCs currently cannot be restored by any means. Recently, genetic aberrations of MM-BMMSCs have been noted, which further impaired their differentiation toward OBs. We hypothesize that the MSCs derived from adipose tissue (ADMSCs) can be used as alternative MSC sources to enhance the pool and function of OBs. Therefore, the purpose of this study was to compare the osteogenesis ability of paired ADMSCs and BMMSCs in MM patients who had completed intensive therapy. Fifteen MM patients who had received bortezomib-based induction and autologous transplantation were enrolled. At the third month after the transplant, the paired ADMSCs and BMMSCs were obtained and cultured. Compared with the BMMSCs, the ADMSCs exhibited a significantly higher expansion capacity (100% vs 13%, respectively; P = .001) and shorter doubling time (28 hours vs 115 hours, respectively; P = .019). After inducing osteogenic differentiation, although the ALP activity did not differ between the ADMSCs and BMMSCs (0.78 U/µg vs 0.74±0.14 U/µg, respectively; P = .834), the ADMSCs still exhibited higher calcium mineralization, which was determined using Alizarin red S (1029 nmole vs 341 nmole, respectively; P = .001) and von Kossa staining (2.6 E+05 µm2 vs 5 E+04 µm2, respectively; P = .042), than the BMMSCs did. Our results suggested that ADMSCs are a feasible MSC source for enhancing the pool and function of OBs in MM patients who have received intensive therapy.

Biological characterization of mesenchymal stem cells from bovine umbilical cord

Mesenchymal stem cells (MSCs) are multi-potential cells that are able to proliferate and differentiate into other cell types. Much research has been done on the MSCs from the umbilical cord (UCMSCs) in human, mice, and avian, but little literature has been published about these cells in big livestock. Here, we choose Luxi cattle as the experimental animal, we describe an external culture of the UCMSCs from it and summarize the biological characteristics of these cells, e.g., morphologic appearance, surface antigens, colony-forming ability, gene expression, and differentiation potential were detected via using immunofluorescence and reverse transcription polymerase chain reaction (RT-PCR). The induced cells, osteoblast, lipoblast, hepatocyte, islet cells, and neurocyte were identified by Alizarin red staining, Oil-red-O staining, Periodic acid-schiff staining, and Dithizone staining and RT-PCR detection for specific genes. Results suggest that biological characteristics of the UCMSCs were similar to those of MSCs previously analyzed. The primary UCMSCs were sub-cultured to passage 32, the UCMSCs expressed gene CD29, CD44, CD73, CD90, and CD166, induced cells illustrated typical staining, and expressed specific genes, which indicate that the UCMSCs could be a novel alternative source of MSCs for experimental and clinical applications.

Comparison of cultured mesenchymal stem cells derived from bone marrow or peripheral blood of rats

Objectives: Mesenchymal stem cells (MSC) are primarily isolated from bone marrow. Peripheral blood is also reported as an alternative source of MSC. This study compared MSC which were isolated and cultured from bone marrow and those from peripheral blood of rats. Methods: MSC from bone marrow and peripheral blood were harvested from 5 male Sprague Dawley rats. After isolation, the cells were grown on tissue culture plates with concentration of 107 cells per well. Observations were conducted to evaluate the attachment of nucleated cells with fibroblast-like morphology. Characterization of MSC was done using reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemistry assay. Results: The mean number of nucleated cells isolated from the bone marrow on day 0 was higher than those isolated from the peripheral blood. Bone marrow MSC with typical fibroblast-like morphology proliferated rapidly and reached 80% confluency on day 14. Subcultures were able to be conducted on day 15 (first passage) and day 28 (second passage). On days 15 and 28, no nucleated cells remained in peripheral blood cultures. Conclusions: Bone marrow derived MSC are more enriched and grow more efficiently than those derived from peripheral blood.

The effect of stem cell from human exfoliated deciduous teeth on T lymphocyte proliferation.

Background:Mesenchymal stem cells (MSC), a specific type of adult tissue stem cell; have the immunosuppressive effects that make them valuable targets for regenerative medicine and treatment of many human illnesses. Hence, MSC have been the subject of numerous studies. The classical source of MSC is adult bone marrow (BM). Due to many shortcomings of harvesting MSC from BM, finding the alternative sources for MSC is an urgent. Stem cells from human exfoliated deciduous teeth (SHED) are relative new MSC populations that fulfill these criteria but their potential immunosuppressive effect has not been studied enough yet. Thus, in this work the effect of SHED on the proliferation of in vitro activated T lymphocytes were explored.Materials and Methods:In this study, both mitogen and alloantigen activated T cells were cultured in the presence of different numbers of SHED. In some co-cultures, activated T cells were in direct contact to MSCs and in other co-cultures; they were separated from SHED by a permeable membrane. In all co-cultures, the proliferation of T cells was measured by ELISA Bromodeoxyuridine proliferation assay.Results:In general, our results showed that SHED significantly suppress the proliferation of activated T cells in a dose-dependent manner. Moreover, the suppression was slightly stronger when MSCs were in physical contact to activated T cells.Conclusion:This study showed that SHED likewise other MSC populations can suppress the activation of T lymphocytes, which can be used instead of BM derived MSCs in many investigational and clinical applications.

The Immunosupressive Effect Of Wharton Jerry -Derived Mesenchymal stem Cells In vitro

BackgroundHuman mesenchymal stem cells (MSCs) can be isolated from several tissues, including bone marrow, umbilical cord, placenta, and adipose tissue. In recent reports, MSCs have the ability to migrate to inflammatory tissues and suppress adverse immune reactions. In fact, BM-derived MSCs are already applied for the patients with acute graft versus host disease (aGVHD) with promising efficacy. To avoid the potential risk of BM harvest, we intended to use umbilical cord Wharton's jelly (WJ) as an alternative source for MSCs. However, the mechanism by which WJ-MSCs exert their immunosuppressive effects is not completely understood. MethodsUmbilical cord was collected after informed consent from the mother. WJ-MSCs were isolated by explants method and in part, continued to culture for further expansion by passage 3 (P3). Otherwise, the remaining WJ-MSCs were cryopreserved in P1. Mixed lymphocyte reaction (MLR) was performed on WJ-MSCs or control. CFSE-labeled human peripheral blood (PB) or cord blood(CB) derived mononuclear cells (MNCs) as the responder cells were mixed with irradiated (50Gy) human CB-derived dendrite cells as stimulator cells at a 10:1 ratio, in the presence of low-dose anti-CD3 antibody. CFSE-labeled responder cells were analyzed by FACS. ResultsWJ-MSCs corresponded to the minimal criteria of MSCs defined by the International Society for Cell Therapy. Thus WJ-MSCs showed spindle-shaped plastic adherent with positive CD105, CD73, CD90, and negative for CD45, CD34, CD14, CD19, and HLA -DR. They also have the ability of differentiation into osteoblasts, adipocytes, chondroblasts in vitro. In MLR, WJ-MSCs efficiently inhibited the responder T cells derived from the same donor of WJ-MSCs, triggered by autologous or allogeneic dendritic cells (DC)(Autologous and allogeneic MLR). Moreover, 3rd party-derived WJ-MSCs also strongly suppressed allogeneic responder T cells proliferation triggered by allogeneic DC(3rd party MLR) (n=3). And WJ-MSCs could inhibit the proliferation of T cells upon PHA-L stimulation. In order to study whether the inhibitory effects of WJ-MSCs on MLR requires the cell to cell contact or not, we next performed MLR on separated WJ-MSCs using by transwell. We observed the inhibitory effect of WJ-MSCs on MLR, although the immunosuppressive effect of the separated WJ-MSCs was much less than that of cell to cell contact. The supernatants of WJ-MSCs, which were derived from 80% confluent WJ-MSCs indicated the mild inhibitory effects on MLR. These inhibitory effects of the WJ-MSCs on MLR were reversed by the addition of indoleamine 2,3-dioxygenase (IDO) inhibitor, 1-methyltryptophan (1-MT) , in a dose dependent manner as shown in Figure 1 (n=3). We also studied the influence of the passage of WJ-MSCs on MLR. The inhibitory effect was comparable among the early passage (∼P3) and long-term passage (P10), although the long-term passage did not show the osteogenic differentiation. DiscussionWe demonstrated WJ-MSCs have the immunosuppressive effect on the stimulated T cells in autologous, allogeneic and 3rd party MLR setting, indicating that immunosuppressive effect of WJ-MSCs is not restricted by MHC. Soluble IDO may play an important role in immunosuppressive effect of MLR, although we need to detect the additional factors especially in cell to cell contact between WJ-MSCs and the responder T cells. Conclusively, WJ-MSCs may be a feasible alternative non-invasive source of BM-MSCs for GVHD treatment and WJ-MSCs banking can be considered for the prompt clinical applications.▪ Disclosures:No relevant conflicts of interest to declare

A novel strategy of spine defect repair with a degradable bioactive scaffold preloaded with adipose-derived stromal cells

Background contextAlthough the use of mesenchymal stem cells (MSC) with scaffolds for bone repair has been considered an effective method, the interactions between implanted materials and bone tissues have not been fully elucidated. At some specific sites, such as the vertebral body (VB) of the spine, the process of bone repair with implanted biomaterials is rarely reported. Recently, adipose tissue was found to be an alternative source of MSC besides bone marrow. However, the strategy of using adipose-derived stromal (ADS) cells with bioactive scaffold for the repair of spinal bone defects has seldom been studied. PurposeTo use a sintered poly(lactide-co-glycolide) acid (PLGA) microspheres scaffold seeded with induced rat ADS cells to repair a bone defect of the VB in a rat model. Study designBasic science and laboratory study. MethodsA sintered porous microspheres scaffold was manufactured by PLGA. ADS cells were isolated from Fischer 344 rats and then induced by osteogenic medium with growth and differentiation factor 5 (GDF5) in vitro. Before implantation, cells were cultured with inductive media for 2 weeks as a monolayer situation and 1 more week on a PLGA scaffold as a three-dimensional structure. These assembled bioactive scaffolds then were implanted in lumbar VB bone defects in Fischer 344 rats. The ex vivo differentiation of the cells was confirmed by von Kossa staining and real-time polymerase chain reaction. The performance of cells on the scaffold was detected by scanning electron microscopy and (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. In vivo bone formation was quantitatively measured by computed tomography study. And the effect of tissue repair was also evaluated by histological studies. ResultsProliferation and differentiation of cells were confirmed before in vivo implantation. Quantification of bone formation in vivo through serial three-dimensional computed tomography images revealed that the VB implanted with GDF5-induced cells demonstrated more bone formation than the control groups. Besides the bone formation period that occurred between 2 and 4 weeks in all groups, a second bone formation period was found to occur only in the groups that received cells with previous induction in vitro. This second period of significant bone formation happened simultaneously with collapsing of the scaffolds. It was then demonstrated histologically that vascularization early in the process and cooperation between host bone and implanted cells accompanied by collapse of the scaffold may be the factors that influence bone formation. This study not only provides a therapeutic strategy of using biomaterial for bone repair in the spine, but also may lead to a technological method for studying the relationship between implanted stem cells and host tissue. ConclusionsAdipose-derived stromal cells maintained in culture on a scaffold and treated with osteogenic induction with growth factor ex vivo could be used to enhance bone repair in vivo.

Human mesenchymal stem cells derived from adipose tissue reduce functional and tissue damage in a rat model of chronic renal failure

Therapeutic approaches for CKD (chronic kidney disease) have been able to reduce proteinuria, but not diminish the disease progression. We have demonstrated beneficial effects by injection of BM (bone marrow)-derived MSCs (mesenchymal stem cells) from healthy donors in a rat model with CKD. However, it has recently been reported that BM-MSCs derived from uraemic patients failed to confer functional protection in a similar model. This suggests that autologous BM-MSCs are not suitable for the treatment of CKD. In the present study, we have explored the potential of MSCs derived from adipose tissue (AD-MSCs) as an alternative source of MSCs for the treatment of CKD. We have isolated AD-MSCs and evaluated their effect on the progression of CKD. Adult male SD (Sprague-Dawley) rats subjected to 5/6 NPX (nephrectomy) received a single intravenous infusion of 0.5×10(6) AD-MSCs or MSC culture medium alone. The therapeutic effect was evaluated by plasma creatinine measurement, structural analysis and angiogenic/epitheliogenic protein expression. AD-MSCs were detected in kidney tissues from NPX animals. This group had a significant reduction in plasma creatinine levels and a lower expression of damage markers ED-1 and α-SMA (α-smooth muscle actin) (P<0.05). In addition, treated rats exhibited a higher level of epitheliogenic [Pax-2 and BMP-7 (bone morphogenetic protein 7)] and angiogenic [VEGF (vascular endothelial growth factor)] proteins. The expression of these biomarkers of regeneration was significantly related to the improvement in renal function. Although many aspects of the cell therapy for CKD remain to be investigated, we provide evidence that AD-MSCs, a less invasive and highly available source of MSCs, exert an important therapeutic effect in this pathology.

The Role of Mesenchymal Stem Cells in Hematopoietic Stem Cell Transplantation: From Bench to Bedsides

Mesenchymal stem cells (MSCs) have been shown to be effective in the management of graft-versus-host disease (GVHD) due to their immunomodulatory effects. In addition to prevention and treatment of GVHD, many studies have demonstrated that MSCs can promote hematopoietic engraftment, accelerate lymphocyte recovery, reduce the risk of graft failure, and repair tissue damage in patients receiving hematopoietic stem cell transplantation (HSCT). Bone marrow (BM) has been considered as the traditional source of MSCs, and most of the knowledge concerning MSCs comes from BM studies. However, BM-derived MSCs have several limitations for their clinical application. Fetal-type MSCs can be isolated easier and proliferate faster in vitro as well as possessing a lower immunogenicity. Therefore, fetal-type MSCs, such as umbilical cord-derived MSCs, represent an excellent alternative source of MSCs. MSCs play multiple important roles in HSCT. Nevertheless, several issues regarding their clinical application remain to be discussed, including the safety of use in humans, the available sources and the convenience of obtaining MSCs, the quality control of in vitro-cultured MSCs and the appropriate cell passages, the optimum cell dose, and the optimum number of infusions. Furthermore, it is important to evaluate whether the rates of cancer relapse and infections increase when using MSCs for GVHD. There are still many questions regarding the clinical application of MSCs to HSCT that need to be answered, and further studies are warranted.

Characterization of the secretome of human tooth germ stem cells (hTGSCs) reveals neuro-protection by fine-tuning micro-environment

Bone-marrow-derived mesenchymal stem cells (MSCs) demonstrate neuro-protective effects in several disease models. By producing growth-factors, cytokines and chemokines, they promote survival of neurons in damaged brain areas. Alternative MSC sources, such as human tooth germ stem cells (hTGSCs), have been investigated for their neuro-protective properties. They ameliorate effects of neuro-toxic agents by paracrine mechanisms, however these secreted bio-active molecules are not yet characterized. Therefore, the current study aimed to provide a detailed analysis of the secretome of hTGSCs. Brain cells were exposed to various toxic materials, including Alzheimer's β-amyloid peptide (β-AP) and 6-hydroxy-dopamine (6-OHDA). When co-cultured with hTGSCs, the activity of a number of anti-oxidant enzymes (catalase, glutathione-s-transferase, glutathione-peroxidase, superoxide-dismutase) was increased and neuronal death/apoptosis was subsequently reduced. The composition of the secreted bio-active materials is influenced by various pre-existing factors such as oxygen and glucose deprivation and the age of cells (passage number). This report reveals for the first time that the neuro-protective secretome of hTGSCs and the micro-environment of cells have a mutual and dynamic impact on one another.

Mesenchymal Stem Cells Derived from Wharton&amp;apos;s Jelly of the Umbilical Cord: Biological Properties and Emerging Clinical Applications

In recent years there seems to be an unbounded interest concerning mesenchymal stem cells (MSCs). This is mainly attributed to their exciting characteristics including long-term ex vivo proliferation, multilineage potential and immunomodulatory properties. In this regard MSCs emerge as attractive candidates for various therapeutic applications. MSCs were originally isolated from the bone marrow (BM) and this population is still considered as the gold standard for MSC applications. Nevertheless the BM has several limitations as source of MSCs, including MSC low frequency in this compartment, the painful isolation procedure and the decline in MSC characteristics with donor's age. Thus, there is accumulating interest in identifying alternative sources for MSCs. To this end MSCs obtained from the Wharton's Jelly (WJ) of umbilical cords (UC) have gained much attention over the last years since they can be easily isolated, without any ethical concerns, from a tissue which is discarded after birth. Furthermore WJ-derived MSCs represent a more primitive population than their adult counterparts, opening new perspectives for cell-based therapies. In this review we will at first give an overview of the biology of WJ-derived UC-MSCs. Then their potential application for the treatment of cancer and immune mediated disorders, such graft versus host disease (GVHD) and systemic lupus erythematosus (SLE) will be discussed, and finally their putative role as feeder layer for ex vivo hematopoietic stem cell (HSC) expansion will be pointed out.

Sox9 Modulates Proliferation and Expression of Osteogenic Markers of Adipose-Derived Stem Cells (ASC)

Background: Mesenchymal stem cells (MSC) are promising tools for tissue-engineering and musculoskeletal regeneration. They reside within various tissues, like adipose tissue, periosteum, synovia, muscle, dermis, blood and bone marrow, latter being the most common tissue used for MSC isolation. A promising alternative source for MSC is adipose tissue due to better availability and higher yield of MSC in comparison to bone marrow. A drawback is the yet fragmentary knowledge of adipose-derived stem cell (ASC) physiology in order to make them a safe tool for in vivo application. Methods/Results: Here, we identified Sox9 as a highly expressed and crucial transcription factor in undifferentiated rat ASC (rASC). In comparison to rat bone marrow-derived stem cells (rBMSC), mRNA and protein levels of Sox9 were significantly higher in rASC. To study the role of Sox9 in detail, we silenced Sox9 with shRNA in rASC and examined proliferation, apoptosis and the expression of osteogenic differentiation markers. Our results clearly point to a difference in the expression profile of osteogenic marker genes between undifferentiated rASC and rBMSC in early passages. Sox9 silencing induced the expression of osteocalcin, Vegfα and Mmp13, and decreased rASC proliferation accompanied with an induction of p21 and cyclin D1 expression and delayed S-phase entry. Conclusions: We suggest a pro-proliferative role for Sox9 in undifferentiated rASC which may explain the higher proliferation rate of rASC compared to rBMSC. Moreover, we propose an osteogenic differentiation delaying role of Sox9 in rASC which suggests that Sox9 expression is needed to maintain rASC in an undifferentiated, proliferative state.

Long-term culture optimization of human omentum fat-derived mesenchymal stem cells

Recent scientific explorations in search of novel sources for autologous transplantation transpired an alternative source of MSCs (mesenchymal stem cells) derived from omentum fat. The scarcity of experimental evidences probing into the biosafety concerns of omentum fat-derived MSC under prolonged culture conditions limits its applicability as an efficient tool in regenerative medicine. This study, thus, aims to optimize human omentum fat-derived MSC in four different media [DMEM (Dulbecco's modified Eagle's medium) LG (low glucose), DMEM KO (knock out), α-MEM (α-minimal essential media) and DMEM F12] in the facets of phenotypic characterization, growth kinetics, differentiation and karyotyping under prolonged culture. The cells exhibited a similarity in expression profile for the majority of markers with evidential variations in certain markers. The relevance of omentum fat-derived MSCs became evident from its triumphant differentiation potential and karyotypic stability substantiated even at later passage. The results obtained from growth curve and PDT (population doubling time) lead to optimization of appropriate media for omentum fat-derived stem cell research, thereby bringing omentum fat into the forefront of regenerative medicine.

Transplantation of cord blood mesenchymal stem cells as spheroids enhances vascularization.

Despite promising results from the therapeutic use of stem cells for treating ischemic diseases, the poor survival of cells transplanted into ischemic regions is one of the major problems that undermine the efficacy of stem cell therapy. Cord blood mononuclear cells (CBMNCs) are an alternative source of mesenchymal stem cells (MSCs) without disadvantages, such as the painful and invasive harvesting procedure, of MSCs derived from bone marrow or adipose tissue. In the present study, we investigated whether the angiogenic efficacy of cord blood mesenchymal stem cells (CBMSCs) can be enhanced by grafting as spheroids in a mouse hindlimb ischemia model. Human CBMSC (hCBMSC) spheroids were prepared by using the hanging-drop method. Mouse hindlimb ischemia was induced by excising the femoral artery and its branches. After surgery, the animals were divided into no-treatment, dissociated hCBMSC, and spheroid hCBMSC groups (n=8 per group) and received corresponding hCBMSC treatments. After surgery, the ischemic hindlimbs were monitored for 4 weeks, and then, the ischemic hindlimb muscles were harvested for histological analysis. Apoptotic signaling, angiogenesis-related signal pathways, and blood vessel formation were investigated in vitro and/or in vivo. The transplantation of hCBMSCs as spheroids into mouse ischemic hindlimbs significantly improved the survival of the transplanted cells by suppressing apoptotic signaling while activating antiapoptotic signaling. Furthermore, the transplantation of hCBMSCs as spheroids significantly increased the number of microvessels and smooth muscle α-actin-positive vessels in the ischemic limbs of mice, and attenuated limb loss and necrosis. Human CBMNC can be considered an alternative source of MSC, and spheroid-based hCBMSC delivery can be considered a simple and effective strategy for enhancing the therapeutic efficacy of hCBMSCs.

Comparative Study of the Biological Characteristics of Mesenchymal Stem Cells from Bone Marrow and Peripheral Blood of Rats

Mesenchymal stem cells (MSCs) from adult exhibit self-renewal and multilineage differentiation capacities, making the MSCs promising candidates for cell therapy and tissue engineering. Although bone marrow (BM) is the most universal source of MSCs, other tissues may also contain MSCs. Peripheral blood (PB), in particular, arises as the most attractive source of MSCs due to easy accessibility and noninvasive procedure. However, it is not certain that PB-MSCs have the equal biological characteristics to those of BM-MSCs. The purpose of this study was to compare the biological characteristics between BM-MSCs and PB-MSCs. We adopted granulocyte colony-stimulating factor combined with CXCR4 antagonist AMD3100 to stimulate MSCs to release into blood circulation of the rats. PB-MSCs were obtained from mobilized PB and expanded in long-term culture. BM-MSCs were isolated from the femur and tibia medullary canal of the same rats by density gradient centrifugation. After cell expansion in vitro, cell surface markers and multipotentiality analysis were performed to identify MSCs. Apoptosis resistance to H(2)O(2)-induced apoptosis, proliferation kinetics, cellular senescence, and karyotype analysis were measured to compare the biological characteristics of PB-MSCs and BM-MSCs. PB-MSCs with the typical adherent fibroblast-like morphology were similar to that of BM-MSCs. Both PB-MSCs and BM-MSCs were positive for CD44 and CD90, and negative for CD34 and CD45. They both exhibited trilineage differentiation potential and expressed lineage-specific genes. Although the BM-MSCs showed stronger osteogenic and adipogenic differentiation, PB-MSCs displayed a more chondrogenic capacity. Further, BM-MSCs have greater proliferation ability. Apoptosis resistance and cellular senescence were similar in MSCs derived from both sources. The results of our study demonstrate that PB-MSCs have similar biological characteristics to those of BM-MSCs despite certain minor differences, suggesting PB as a possible alternative source for MSCs.

Application of human amniotic mesenchymal cells as an allogeneic transplantation cell source in bone regenerative therapy

Autogenous mesenchymal stem cells (MSCs) have therapeutic applications in bone regenerative therapy due to their pluripotency. However, the ability of MSCs to proliferate and differentiate varies between donors. Furthermore, alternative sources of MSCs are required for patients with contraindications to autogenous cell therapy. The aim of this study was to evaluate the potential of mesenchymal cells from the human amniotic membrane (HAM) as a source of cells for allogeneic transplantation in bone regenerative therapy. Cells that retained a proliferative capacity of more than 50 population doubling level were distinguished from other HAM cells as HAMα cells and induced to osteogenic status—their in vivo osteogenesis was subsequently investigated in rats. It was found that HAMα cells were spindle shaped and were positive for MSC markers and negative for hematopoietic stem cell markers. Alkaline phosphatase activity and calcium deposition increased with osteogenic status of HAMα cells. The expression of osteocalcin mRNA was increased in HAMα cells cultured on calcium phosphate scaffolds. Moreover, xenografted HAMα cells remained viable and produced extracellular matrix for several weeks. Thus, this study suggests that human amniotic mesenchymal cells possess osteogenic differentiation potential and could be applied to allogeneic transplantation in bone regenerative therapy.

Characterization and immunosuppressive properties of mesenchymal stem cells from periapical lesions

Mesenchymal stem cells (MSCs) isolated from healthy dental tissues are being investigated as an alternative source of MSCs for the treatment of damaged tissues and inflammatory diseases. Here we investigated whether MSCs from periapical lesions (PL-MSCs) also possess multi-lineage differentiation capacity and immunomodulatory properties. PL-MSCs, isolated by collagenase/DNAse digestion from surgically extracted PLs, were compared with MSCs from non-inflamed dental pulp (DP-MSCs) and dental follicle (DF-MSCs) for their phenotype and multi-potent differentiation potential. The anti-inflammatory and immunomodulatory effects of PL-MSCs were studied in co-culture with peripheral blood mononuclear cells (PB-MNCs) and PL-inflammatory cells (PL-ICs). PL-MSCs were characterized by typical MSCs phenotype, lower clonogenicity and self-renewal rate, compared to DF-MSCs and DP-MSCs. These cells possess the potential to differentiate into adipocyte-, osteoblast- and chondrocyte-like cells in vitro, which differs from that of DP-MSCs and DF-MSCs. PL-MSCs inhibited phytohemaglutinine-induced proliferation of PB-MNCs and production of IL-2, IFNγ and IL-5 in the co-culture, probably via TGF-β-dependent mechanisms. These cells also suppressed the production of IL-1β, IL-6, and TNF-α by PL-ICs via soluble mediators, whereas the suppression of IL-8 production required a direct cell-to-cell contact. The differentiation potential of PL-MSCs and their immunosuppressive/anti-inflammatory properties could be beneficial for the treatment of chronic periodontal diseases.

Isolation and Surface Characterization of Mesenchymal Stem Cells(MSCs) Derived From Term Human Amnion

ObjectiveArticular cartilage damage has a very low spontaneous healing capacity. Emerging evidence suggests human placental membrane is a valuable source of MSCs for cell‐based therapy to promote articular cartilage repair. However, the identification of MSC has been hampered. Thus there is a clear need to develop strategies leading to the identification of potential MSC markers. The objective of this study was to isolate and characterize the cell populations derived from term pregnancy human amnion.MethodsCells were isolated from amnion membranes from patients at gestational weeks 39±1. Flow cytometry analysis was performed with the cells. Direct conjugated Ab used for flow cytometry included anti‐CD44‐FITC, CD73‐FITC, CD105‐FITC, CD45‐PerCP, CD90‐PerCP, CD11b‐PE, CD29‐PE, CD34‐PE. Analysis was performed with FlowJo software.ResultsThe positive markers in isolated cell populations on Day 0 were CD14, CD11b, CD29, CD44, CD45, CD90, CD105. After 10 days culturing in expansion medium, the macrophage marker CD14 disappeared and mesenchymal stem cell markers (CD29, CD44, CD90, CD73, CD105) were demonstrated increased expression levels. Throughout, the cells were negative for markers of developmental stages of blood cells(CD11b, CD45).ConclusionOur results indicate that MSCs can be isolated from the human term placental membranes. The human amniotic MSCs maintained a marker profile similar to the mesenchymal progenitors and could be used for studies as an alternative source of MSCs for further application in cell‐based therapies.