Isolation Of Mesenchymal Stem Cells Research Articles

Isolation and characterization of deer-derived mesenchymal stem cells

Isolation and characterization of deer-derived mesenchymal stem cells

The effects of transplanted mesenchymal stem cells treated with 17-b estradiol on experimental autoimmune encephalomyelitis.

The present study was conducted aimed at exploring the modulatory effects of 17-b estradiol (17-bED) on mesenchymal stem cells (MSCs) in the EAE (experimental autoimmune encephalomyelitis) animal model of multiple sclerosis (MS). Following the isolation of bone marrow-derived MSCs from the bilateral femurs and tibias of the male Wistar rats, the cells were harvested and cultured in the presence of 100nM 17-bED for 24h. EAE was induced in male Wistar rats (8-12weeks old) using guinea pig spinal cord homogenate, in combination with the complete Freund's adjuvant. The MSC therapy was triggered when all of the animals obtained a disability score. The symptoms were monitored on a daily basis throughout the study until the rats were euthanized. The mRNA expression of cytokines, including IL-17, IFN-γ, TNF-α, IL-10, IL-4, and TGF-β together with MMP8 and MMP9 as the family members of matrix metalloproteinases(MMPs) in the brain and spinal cord tissues were examined using real-time PCR. The levels of splenocytes-originated IL-10 and IFN-γ cytokines were also measured by ELISA. The MTT-based research findings showed that the infiltration of lymphocytes into the spleen decreased considerably. It was also observed that the mRNA expression of proinflammatory cytokines decreased significantly, while the mRNA levels of anti-inflammatory cytokines increased remarkably. It was also found that the mRNA levels of the examined matrix metalloproteinases(MMP8 and MMP9) were downregulated significantly. The findings of the present study indicated that the administration of 17-bED enhanced the efficacy of MSCs transplantation and modulated immune responses relatively in the EAE model, via the regulation of either pro- or anti-inflammatory cytokines and matrix metalloproteinases.

The Effect of Mesenchymal Stem Cells Pulsed with 17 Beta-Estradiol in an Ameliorating Rat Model of Ulcerative Colitis

Background: Among the available supplements, 17 β-estradiol (E2) has strong proliferative effects on mesenchymal stem cells (MSCs). Objectives: The current study aimed at evaluating the effects of E2-primed MSCs therapy in a rat model of ulcerative colitis. Methods: After the isolation of MSCs, cells were co-cultured with E2 for 24 hours. Colitis was induced by acetic acid in four groups; the control colitis, MSCs-treated, E2-primed MSCs-treated (E2-MSCs), and normal. MSCs and E2-MSCs were injected into the peritoneum in two distinct groups. After 10 days, the rats were evaluated for the level of nitric oxide, malondialdehyde, myeloperoxidase, total protein, and proinflammatory cytokines (IL-6, IL-1β, and TNF-α) in colonic homogenates. Results: The cell therapy with E2-MSCs in rats with colitis had a more desirable outcome; indeed, it resulted in a favorable regression in clinical score and inflammatory profile in rats with colitis than the MSCs-treated. The levels of myeloperoxidase, nitric oxide, and malondialdehyde were significantly diminished and, conversely, the total protein levels were significantly increased in rats with ulcerative colitis receiving E2-MSCs compared to rats with colitis receiving untreated MSCs. On the same side, the level of proinflammatory cytokines reduced significantly in E2-MSCs rats than the MSCs group. Conclusions: E2-MSCs decrease colonic damage significantly, which is probably associated with the prominent reduction of lipid peroxidation, neutrophil infiltration, and the levels of proinflammatory cytokines in the colonic tissue. Therefore, it seems that E2 effectively surmounts the limitations of MSC application and makes it an ideal choice for cell therapy in inflammatory diseases such as ulcerative colitis.

Isolation of umbilical cord mesenchymal stem cells using human blood derivatives accompanied with explant method.

Mesenchymal stem cells (MSCs) are multipotent cells which can be isolated from many sources including umbilical cord. Isolation protocols are depended on either explant or enzymatic methods. Although fetal bovine serum (FBS) is used as a supplement in isolation and expansion of MSCs, human blood derivatives such as cord blood serum (CBS) and platelet lysate (PL) are attractive substitutes of FBS which overcome impediments of using FBS in clinical setups. Here we compared the effect of using CBS, PL and FBS supplemented media in isolation of umbilical cord tissue derived MSCs by using explant method. To do that we cultured umbilical cord tissue explant in either CBS or PL or FBS supplemented media. Isolated cells were quantified, their morphology was assessed and Cells in passage 3 were characterized based on their immunophenotyping and their potential for differentiation into adipocytes and osteocytes. Moreover, proliferation of cells was assessed by crystal violet staining. All the three media succeeded to isolate MSCs and maintain their stemness characteristics. However, the highest number of isolated cells were obtained using CBS, ~10-fold more than FBS, while the number of isolated cells obtained using PL was ~2-fold more than FBS. Moreover, crystal violet showed that both PL and CBS promote proliferation of MSCs more than FBS. Our data suggest that, although all supplements maintain stemness characteristics of MSCs when used to isolate those cells by explant method, using human blood derived supplements is more effective than FBS. In the same context CBS is more effective than PL.

45: The Effect of Transplantation of Undifferentiated Mesenchymal Stem Cells in Experimental Model of Myocardial Infarction: Preliminary Results

Introduction: Cardiovascular diseases like ischemic myocardial infarction remain worldwide with high lethality and mortality beside the extended researches for pharmaceutical therapies. Isolation of mesenchymal stem cells (ADSCs) from adipose tissue created a new spectrum of research. ADSCs are now known to be able to differentiate to myocardial cells and create cardiac tissue. In our protocol we use the genetic factors GATA-4 and nkx2,5 which are associated with the regeneration and differentiation of the myocardium respectively. Methods: We used 17 adult Wistar rats of conventional microbiological status (15 females – receivers and 2 males - donors) weighting 200-250gr. The adipose tissue was collected from the male donors and was immediately transferred to the laboratory at 4°C. The female-receivers were randomly allocated in 5 groups (3 animals per group). On Day -7 (7 days pre-operatively) we performed a SPECT–CT with injection of 99mTc-sestamibi to all female animals. On Day 0 (operation day) all groups underwent left thoracotomy and - besides sham operated - ligation of LAD for 45min with ECG monitoring confirming ischemia. Control 1: injection of N/S intramyocardially and removal of ligation. Control 2: injection of N/S intramyocardially without removal of ligation. Sham: no ligation. Experimental 1: injection of ADSCs intramyocardially and removal of ligation. Experimental 2: injection of ADSCs intramyocardially without removal of ligation. On Day +7 and Day +14 (7 and 14 days post-operatively) the animals that survived underwent new SPECT-CT followed by euthanasia on Day +16 (16 days postoperatively) together with blood sampling and heart harvesting for histological and immunohistochemical evaluation. Results: ADSCs were successfully engrafted into the myocardium and had beneficial effect on the ischemic myocardial area of the experimental groups with regeneration and increase of contractility especially in the 14th postoperative day compared with the control groups. Specifically imaging with SPECT-CT revealed significant absorption of 99Tc and viability of the myocardium on postoperative Day 14 in the experimental groups. The expression of GATA-4, nkx2,5 and IL-6 was significantly upregulated compared with the control groups. Conclusion: ADSCs are definitely the therapeutic approach of the future and GATA-4 as nkx2,5 are the genetic factors that are leading to an extended regeneration of the ischemic myocardium. The promising results so far are leading us to increase the number of animals in our protocol to further evaluate the clinical significance of ADSCs transplantation in cases of acute myocardial infarction.

Xenogen-free isolation and culture of human adipose mesenchymal stem cells.

Adipose-derived Stem Cells (ASCs) present great potential for reconstructive procedures. Currently, isolation by enzyme digestion and culturing using xenogenic substances remain the gold standard, impairing clinical use. Abdominal lipo-aspirate and blood samples were obtained from healthy patients. A novel mechanical isolation method for ASCs was compared to (the standard) collagenase digestion. ASCs are examined by flowcytometry and multilineage differentiation assays. Cell cultures were performed without xenogenic or toxic substances, using autologous plasma extracted from peripheral blood. After eGFP-transfection, an in vivo differentiation assay was performed. Mechanical isolation is more successful in isolating CD34+/CD31-/CD45-/CD13+/CD73+/CD146- ASCs from lipo-aspirate than isolation via collagenase digestion (p <0 .05). ASCs display multilineage differentiation potential in vitro. Autologous plasma is a valid additive for ASCs culturing. eGFP-ASCs, retrieved after 3 months in vivo, differentiated in adipocytes and endothelial cells. A practical method for human ASC isolation and culturing from abdominal lipo-aspirate, without the addition of xenogenic substances, is described. The mechanical protocol is more successful than the current gold standard protocol of enzyme digestion. These results are important in the translation of laboratory-based cell cultures to clinical reconstructive and aesthetic applications.

Possible Renal Repairing Mechanisms of Mesenchymal Stem Cells in Cyclosporine-Mediated Nephrotoxicity: Endothelial Viability and Hemodynamics

BACKGROUND: Stem cell-based therapy may represent the first realistic option for tissue repairing and regeneration. Mesenchymal stem cells (MSCs) are proved to be involved in the regeneration of many tissues which are subjected to different types of injury. Cyclosporine (CsA) in spite of its use as immune suppressive drug during kidney transplantation, it was considered as important model of nephropathy specially, during long-term administration.METHODS: Isolation and preparation of MSCs using Dulbecco's modified Eagle's medium (DMEM), in vitro differentiation through adipogenesis chondrogenesis and osteogenesis was confirmed by using Alizarin Red S stain, Oil Red O stain and Alcian Blue stain, respectively, characterization using flow cytometry technique to detect cluster of differentiation (CD)34, CD44 and CD105 surface markers and homing of MSCs using polymerase chain reaction (PCR) Sry gene assay, were executed. Serum levels of vascular endothelial growth factor (VEGF), endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) were quantified using enzyme-linked immunosorbent assay (ELISA) kits based on the principle of double-antibody sandwich technique. The structural organization of kidney tissue was examined using histological procedures.RESULTS: Single intravenous dose of MSCs is capable to boost kidney repairment process as indicated by the significant decrease in serum ET-1 level paralleled by significant increase in VEGF and eNOS. Moreover, histological findings revealed the improvement of focal hemorrhage in between the degenerated tubules, congestion in the cortical blood vessels, vacuolization in the endothelial cells lining the glomerular tufts and focal perivascular inflammatory cells aggregation.CONCLUSION: This study demonstrated the favorable influence of MSCs in repressing cyclosporine-induced nephropathy in rats. This could be achieved through angiogenic and anti-arteriolopathic mechanisms.KEYWORDS: angiogenesis, cyclosporine, endothelin-1, MSCs, nephropathy

Biological Characteristics and Multilineage Differentiation of Kidney Mesenchymal Stem Cells Derived from the Tibetan Mastiff

Of all the significant researches that have taken place in isolation, culture and characterization of mesenchymal stem cells (MSCs), the field of kidney-derived mesenchymal stem cells (KMSCs) in Tibetan mastiff is still a blank. Therefore, the purpose of this study is to isolate, culture and characterize the Tibetan mastiff KMSCs. The KMSCs were successfully isolated from one-day year old Tibetan mastiff kidney, cultured for 16 passages and distinguished by two methods: immunofluorescence staining and RT-PCR. The Tibetan mastiff KMSCs expressed specific surface marker genes (VIM, CD44, FN1, CD90, CD109, CD73, FN1) and kidney marker gene PAX2. The proliferation ability of Tibetan mastiff KMSCs was measured through cell count and clonality. Furthermore, cells differentiated into different cell types (hepatocellular cells, osteogenic cells, adipogenic cells and chondrogenic cells) under special induced medium, and the marker genes of induced cells were identified with Immunofluorescence staining and RT-PCR. All of these results indicated that the Tibetan mastiff KMSCs were obtained successfully, which possessed certain characteristics of multipotent stem cells. Therefore, MSCs in Tibetan mastiff kidney hold potential for clinical applications for regenerative therapy and their further studies are waiting to be required to investigate their functions.

The Subacromial Bursa is a Viable Source of Autologous Mesenchymal Stem Cells for Rotator Cuff Repair

Objectives: Chronic rotator cuff tears still represent a significant source of morbidity and functional decline in the general population. The purpose of this study was to establish protocols for isolation and expansion of bursa-derived mesenchymal stem cells (BDSCs) and to evaluate their differentiation capacity, including tenogenesis. We hypothesized that BDSCs would be capable of multilineage differentiation (including tenogenesis) and represent an important source for autologous stem cells for patients undergoing rotator cuff repair. Methods: After IRB approval, 10 patients (ages 43-65 years) scheduled to undergo arthroscopic repair for chronic rotator cuff tears were enrolled. During diagnostic arthroscopy, subacromial bursa tissue was harvested using an arthroscopic shaver and collected by attaching the outflow tubing to a specialized specimen cup. Tissue specimens were transported to our laboratory for analysis. BDSCs were isolated via adherent culture and plated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS). Chondrogenic, adipogenic, and osteogenic induction media were used to induce differentiation. Tenogenic induction was performed using DMEM supplemented with varying concentrations of BMP-12, ascorbic acid, and human tenocyte-conditioned media. Alcian Blue staining was used to evaluate chondrogenesis, Oil Red O staining for adipogenesis, and Alkaline Phosphatase staining for osteogenesis. Gene expression markers for adipogenesis (ADIPOQ, FABP4, PPARγ), chondrogenesis (COL2A1 and SOX5), and osteogenesis (osteocalcin, osterix), along with primary antibodies to tenogenic markers (scleraxis, tenomodulin), were used to verify each cell lineage. Results: BDSCs isolated by adherent culture without collagen exhibited a spindle-shaped morphology characteristic of mesenchymal stem cells (MSCs), formed colonies, and demonstrated great expandability for six to eight passages without morphology changes (Figure 1A). After 3 weeks of culture, 95% (p&lt;0.0001) of the BDSCs expressed the MSC surface marker CD90 and were negative for non-MSC cell markers (CD45, CD146, CD31, and CD34) (Figure 1B and C). These BDSCs demonstrated a capacity for adipogenesis (positive Oil Red O staining, ADIPOQ, FABP4, PPARγ expression), osteogenesis (positive alkaline phosphatase staining, positive osteocalcin and scleraxis expression), chondrogenesis (positive Alcian Blue staining, positive COL2A1 and SOX5 expression), and tenogenesis (scleraxis and tenomodulin expression) (Figure 2). The results indicate that BDSCs are multipotent as evidenced by their differentiation into fat, bone, cartilage, and tendon cells. For tenogenesis, we found that 7-day incubation in DMEM supplemented with 100 ng/mL of BMP-12 and 50 μg/mL of ascorbic acid produced superior tenogenic induction. These BDSCs adapted an elongated morphology combined with the expression of both scleraxis and tenomodulin - a unique characteristic of native tenocytes. Conclusion: Our results demonstrate that subacromial bursa represents a viable source of mesenchymal stem cells. We developed a reliable protocol for isolation of BDSCs from patient bursa samples. We show that BDSCs in the presence of BMP-12 and ascorbic acid can differentiate toward a tenogenic lineage. Our work provides strong evidence that BDSCs may be a potent tool for cellular therapy and may benefit future patients who undergo surgical repair of chronic rotator cuff tears. [Figure: see text][Figure: see text]

Beyond a “Pinch of this and a Handful of that:” Using Manufacturing Standards for the Isolation of Human Mesenchymal Stem Cells From Adipose Tissue- A Novel Point-of-Care Device

Beyond a “Pinch of this and a Handful of that:” Using Manufacturing Standards for the Isolation of Human Mesenchymal Stem Cells From Adipose Tissue- A Novel Point-of-Care Device

Влияние облучения в сверхвысоких дозах на криоконсервированные мезенхимальные стволовые клетки: двунитевые разрывы ДНК и пролиферативная активность

Purpose: To conduct a comparative assessment of the effect of irradiation of human mesenchymal stem cells (MSC)at ultrahigh doses at liquid nitrogen temperature (–196 °C) and room temperature (+22 °C) on the yield of “residual” DNA double-strand breaks (DSB) and proliferative activity of thawed MSC.&#x0D; Material and methods: Isolation and cultivation of MSC was carried out according to standard methods. Dimethyl sulfoxide (DMSO) at a final concentration of 10 % was used for cells cryopreservation. The cells were irradiated with bremsstrahlung photon radiation with photon nominal energy 5 MeV, the accelerator UELR-10-100-T-100 (Russia). Cells were irradiated at the doses of 50 and 500 Gy at a temperature of 22 °C and –196 °C. The yield of “residual” DNA DSB was assessed using an immunocytochemical analysis of the foci of the protein-marker DSB – γH2AX. To assess the proliferative activity, the number of Ki67 (protein marker of cell proliferation) of positive cells was analyzed.&#x0D; Results: The results of γH2AX foci assessment in MSCs after 48 hours irradiation at a dose of 50 Gy showed that the number of “residual” γH2AX foci in the MSC nuclei irradiated at 22 °C is about 3.2 times (p = 0.0002) higher than in the MSC nuclei irradiated at –196 °C. Analysis of the proliferative activity of cells using the molecular marker of cell proliferation of the Ki67 protein showed that cells irradiated at a dose of 50 Gy at a temperature of 22 °C, completely lost their ability to proliferate. The proliferative activity of cells irradiated at the same dose, but at a temperature of –196 °C, is significantly reduced, but some of the cells (3.5 ± 1.1 %) still retain the ability to proliferate. After irradiation with a dose of 500 Gy at –196 °C, the cells completely lose their ability to proliferate, but partially retain the ability to adhere. The integral fluorescence conjugated with the flurochrome of antibodies to γH2AX of MSC nuclei irradiated at a dose of 500 Gy at a temperature of –196 °C is 1.8 times lower than that of nuclei irradiated at a temperature of 22 °C.&#x0D; Conclusion: The results of the studies indicate that cryopreserved MSCs irradiated at liquid nitrogen temperature (–196 °C) in a preservation medium containing 10 % DMSO can tolerate the effects of ionizing radiation in large doses (up to 50 Gy). However, there is a rather high yield of “residual” DSB DNA and a very low proliferative activity, which makes them unsuitable in clinical practice. It seems promising to use a quantitative analysis of γH2AX foci to assess genome damage and the functional state of cells irradiated in a cryopreserved state.

Isolation of canine adipose-derived mesenchymal stem cells from falciform tissue obtained via laparoscopic morcellation: A pilot study.

To evaluate the feasibility of stem cell isolation from falciform fat harvested via laparoscopic morcellation. Pilot study. Eleven client-owned dogs. Falciform was harvested traditionally via laparotomy and laparoscopically via tissue morcellation. Harvested tissue was processed with a commercially available adipose tissue dissociation kit to obtain a stromal vascular fraction (SVF). Cells were subsequently labeled for CD90, CD45, and CD44 cell surface antigens by using magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting flow cytometry. CD90+ cells were quantitated, and their viability was assessed with a hemocytometer and a trypan blue exclusion test of cell viability. No perioperative complications occurred in dogs undergoing laparoscopic morcellation. Laparoscopically and traditionally harvested samples yielded an average of 0.39 (±0.1) × 106 and 0.33 (±0.1) × 106 CD90+ cells, respectively, per 10 million SVF cells. CD90+ cell viability after MACS was 89% (±11%) for morcellated and 86% (±7%) for traditionally harvested samples. Neither CD90+ cell quantity nor viability was different between samples obtained via traditional laparotomy vs laparoscopic morcellation (P = .38 and P = .63, respectively). Populations of CD90+ cells isolated with each harvest technique had similar CD44 and CD45 expression profiles. Viable populations of CD90+ cells with similar CD44/CD45 expression profiles were isolated from laparoscopically morcellated and traditionally harvested falciform tissue. No appreciable morbidity was associated with laparoscopic falciform morcellation. Laparoscopic morcellation is a safe and effective minimally invasive approach to falciform tissue harvest for adipose-derived mesenchymal stem cell isolation.

Comparison of different methods for the isolation and purification of rat nucleus pulposus-derived mesenchymal stem cells

ABSTRACT Purpose Recently, nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have been identified and have shown good prospects for the repair of degenerative intervertebral discs. However, there is no consensus about the methods for the isolation and purification of NPMSCs. Therefore, a reliable and efficient isolation and purification method is potentially needed. We aimed to compare different methods and to identify an optimal method for isolating and purifying NPMSCs. Methods NPMSCs were isolated and purified using two common methods (a low-density culture (LD) method and a mesenchymal stem cell complete medium culture (MSC-CM) method) and two novel methods (a cloning cylinder (CC) method and a combination of the CC and MSC-CM methods (MSC-CM+CC)). The morphology, MSC-specific surface markers (CD44, CD73, CD90, CD105, CD34 and HLA-DR), multiple-lineage differentiation potential, colony formation ability, and stemness gene (Oct4, Nanog, and Sox2) expression were evaluated and compared. Results NPMSCs isolated from nucleus pulposus (NP) tissues via the four methods met the criteria stated by the International Society of Cell Therapy (ISCT) for MSCs, including adherent growth ability, MSC-specific surface antigen expression, and multi-lineage differentiation potential. In particular, the MSC-CM+CC method yielded a relatively higher quality of NPMSCs in terms of cell surface markers, multiple-lineage differentiation potential, colony formation ability, and stemness gene expression. Conclusions Our results indicated that NPMSCs can be obtained via all four methods and that the MSC-CM+CC method is more reliable and efficient than the other three methods. The findings from this study provide an alternative option for isolating and purifying NPMSCs.

Isolation, Characterization, and Agent-Based Modeling of Mesenchymal Stem Cells in a Bio-construct for Myocardial Regeneration Scaffold Design

Regenerative medicine involves methods to control and modify normal tissue repair processes. Polymer and cell constructs are under research to create tissue that replaces the affected area in cardiac tissue after myocardial infarction (MI). The aim of the present study is to evaluate the behavior of differentiated and undifferentiated mesenchymal stem cells (MSCs) in vitro and in silico and to compare the results that both offer when it comes to the design process of biodevices for the treatment of infarcted myocardium in biomodels. To assess in vitro behavior, MSCs are isolated from rat bone marrow and seeded undifferentiated and differentiated in multiple scaffolds of a gelled biomaterial. Subsequently, cell behavior is evaluated by trypan blue and fluorescence microscopy, which showed that the cells presented high viability and low cell migration in the biomaterial. An agent-based model intended to reproduce as closely as possible the behavior of individual MSCs by simulating cellular-level processes was developed, where the in vitro results are used to identify parameters in the agent-based model that is developed, and which simulates cellular-level processes: Apoptosis, differentiation, proliferation, and migration. Thanks to the results obtained, suggestions for good results in the design and fabrication of the proposed scaffolds and how an agent-based model can be helpful for testing hypothesis are presented in the discussion. It is concluded that assessment of cell behavior through the observation of viability, proliferation, migration, inflammation reduction, and spatial composition in vitro and in silico, represents an appropriate strategy for scaffold engineering.

Impact of HIV/simian immunodeficiency virus infection and viral proteins on adipose tissue fibrosis and adipogenesis

HIV-infected patients receiving antiretroviral treatment (ART) often present adipose tissue accumulation and/or redistribution. adipose tissue has been shown to be an HIV/SIV reservoir and viral proteins as Tat or Nef can be released by infected immune cells and exert a bystander effect on adipocytes or precursors. Our aim was to demonstrate that SIV/HIV infection per se could alter adipose tissue structure and/or function. Morphological and functional alterations of subcutaneous (SCAT) and visceral adipose tissue (VAT) were studied in SIV-infected macaques and HIV-infected ART-controlled patients. To analyze the effect of Tat or Nef, we used human adipose stem cells (ASCs) issued from healthy donors, and analyzed adipogenesis and extracellular matrix component production using two dimensional (2D) and three-dimensional (3D) culture models. Adipocyte size and index of fibrosis were determined on Sirius red-stained adipose tissue samples. Proliferating and adipocyte 2D-differentiating or 3D-differentiating ASCs were treated chronically with Tat or Nef. mRNA, protein expression and secretion were examined by RT-PCR, western-blot and ELISA. SCAT and VAT from SIV-infected macaques displayed small adipocytes, decreased adipogenesis and severe fibrosis with collagen deposition. SCAT and VAT from HIV-infected ART-controlled patients presented similar alterations. In vitro, Tat and/or Nef induced a profibrotic phenotype in undifferentiated ASCs and altered adipogenesis and collagen production in adipocyte-differentiating ASCs. We demonstrate here a specific role for HIV/SIV infection per se on adipose tissue fibrosis and adipogenesis, probably through the release of viral proteins, which could be involved in adipose tissue dysfunction contributing to cardiometabolic alterations of HIV-infected individuals.

Bench-to-bedside optimization of mesenchymal stem cell isolation, processing, and expansion for in vivo administration.

Aim: In this study, we aimed at identifying the optimal conditions for isolation, processing and expansion of mesenchymal stem cells (MSCs). Methods: Porcine bone marrow was obtained from either small- or large-volume bone marrow aspirate (BMA). Next, three BMA processing methods were compared. Finally, the best condition was selected from various culture parameters, including basal media, supplementation and seeding density. Results: Our results demonstrate that a small-volume BMA and direct plating yields significantly higher concentration of MSCs. Basal media supplementation with 10% platelet lysate and seeding density of 1000cells/cm2 can generate large numbers of multipotent MSCs with augmented function and low population doublings. Conclusion: This work provides guidance for preparation of robust MSCs for future clinical trials.

Isolation and culture of adipose-derived mesenchymal stem cells from inbreed line miniature pig of Wuzhishan and their biological characteristics

To establish a method for isolation, culture and identification of adipose-derived mesenchymal stem cells (ASCs) from the inbreed line miniature pig of Wuzhishan (ILMW). Methods: A total of 100 g adipose tissues were obtained from subcutaneous tissues of neck in six-month old healthy ILMW (3 samples, male). ASCs from ILMW (ILMW-ASCs) were isolated from adipose tissues through 0.1% collagenase digestion. The cells at the 3rd, 5th, 8th, 13th passages were collected. Cell morphology, size, phenotype, cell cycle, and apoptosis were monitored. Cell differentiation was induced and cell proliferation curve was drawn. Results: The ILMW-ASCs, fibroblast-like or whirlpool-like, began the adherence at 36 h and entered a logarithmic phase in the 5th day. Eighty percent of them were fused in the 7th day. The average diameter and volume of ILMW-ASCs were (17.00±0.54) µm and (2.58±0.24)×10-9 L, respectively. The expressions of CD29, CD44 and CD90 were positive, and there was no significant difference between the different passages (all P>0.05). The expressions of CD45, CD8a and HLA-DR were increased with the increase in passages after the 3th passage (all P<0.05). The adipogenic induction of ILMW-ASCs was observed by positive oil red O staining, and the osteogenic induction of ILMW-ASCs was determined by positive alizarin red staining. Apoptosis and senescence occurred in the 13 passage of ILMW-ASCs, and the proportion of S phase of cell cycle was lower than that in lower passages (all P<0.05). Conclusion: ILMW-ASCs are one of the best choice for porcine ASCs, which might provide a source of candidate stem cells for therapy of large animal disease models and tissue or organ repairment.

A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Research and development of osteogenic differentiation of bone marrow mesenchymal stem cells

Supercritical large area or large segmental bone defects are still a clinical problem. Researchers are committed to the development of artificial bone materials, but in order to solve the problem of poor bone formation of artificial bone materials, people are paying more and more attention to application of bone marrow mesenchymal stem cells in bone tissue engineering. In this review, bone marrow mesenchymal stem cells, osteogenic differentiation, osteoblasts cells, clinical application were used as keywords to search CNKI database, Wanfang database, Weipu database and PUBMED database by computer. The isolation and culture of bone marrow mesenchymal stem cells, bone marrow mesenchymal stem cell identification method, osteogenic induction method, osteogenic differentiation identification and clinical applicationt were comprehensively summarized in order to provide a theoretical basis for its use as a seed cell in the treatment of bone tissue diseases. Scholars have preliminarily studied the treatment of bone and cartilage defects, osteoarthritis, femoral head necrosis and other diseases with bone marrow mesenchymal stem cells combined with transplantation, and obtained good clinical efficacy.However, bone marrow mesenchymal stem cells have certain advantages and disadvantages, and further clinical studies and long-term efficacy verification are needed.

IL‐7 overexpression enhances therapeutic potential of rat bone marrow mesenchymal stem cells for diabetic wounds

This study was aimed to enhance the healing potential of rat bone marrow mesenchymal stem cells against chronic diabetic wounds through interleukin-7 (IL-7) transfection. IL-7 plays an important role in wound healing and acts as a survival factor in some cell types. This study involves isolation, propagation, and characterization of mesenchymal stem cells (MSCs) and their modification with IL-7 gene via retroviral transfection. Transfected MSCs were assessed for their effect on angiogenic genes by qPCR. Wound healing potential of transfected MSCs was analyzed by scratch assay in vitro and by transplanting these cells in rat diabetic wound models in vivo. Wound area was measured for a period of 15 days and subsequent histological analysis was performed. qPCR results showed increased expression of IL-7 gene (p ≤ 0.05) and also principal angiogenic genes, vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), VEGF receptor 1 (FLT-1), and VEGF receptor 2 (FLK-1) (p ≤ 0.05). Neuropilin-1 (NRP-1) did not show any significant change. In vitro analysis of IL-7 MSCs showed intense cell-cell connections and tube formation as compared to the normal MSCs. Rate of wound closure was more (p ≤ 0.001) in case of diabetic group transplanted with IL-7 MSCs. Histological examination revealed enhanced vascular supply in skin tissues of diabetic animals transplanted with IL-7 transfected MSCs as compared to normal MSCs. Immunohistochemical results showed significantly higher expression of IL-7 (p ≤ 0.001) and α-smooth muscle actin(p ≤ 0.001) in the tissue sections of IL-7 transfected group as compared to normal MSCs and the diabetic control group; the latter indicates increase in the number of blood vessels. It is concluded from this study that IL-7 overexpression in MSCs can enhance the healing potential of MSCs and aid in wound closure in diabetic animals through the induction of angiogenic genes.