Xeno-free proliferation of human bone marrow mesenchymal stem cells

The proliferation of human bone marrow mesenchymal stem cells (MSCs) employing xeno-free materials not containing fetal calf serum (FCS) and porcine trypsin was investigated for the regenerative medicine of cartilage using MSCs. Four sequential subcultivations of MSCs using a medium containing 10% FCS and recombinant trypsin (TrypLESelect™) resulted in cell growth comparable to that with porcine trypsin. There was no apparent difference in the cell growth and morphology between two kinds of MSC stored in liquid nitrogen using 10% FCS plus DMSO or serum-free TC protector™. MSCs were isolated from human bone marrow cells, stored in liquid nitrogen, and sequentially subcultivated four times employing conventional materials that included FCS, porcine trypsin, and DMSO, or xeno-free materials that included serum-free medium (MesenCult-XF™), TC protector™ and TrypLESelect™. Cells in the culture using the xeno-free materials maintained typical fibroblast-like morphology and grew more rapidly than the cells in the culture using the conventional materials, while the cell surface markers of MSCs (CD90 and CD166) were well maintained in both cultures. Chondrogenic pellet cultures were carried out using these subcultivated cells and a medium containing TGFβ3 and IGF1. The pellet culture using cells grown with the xeno-free materials showed an apparently higher gene expression of aggrecan, a chondrocyte marker, than the pellet culture using cells grown with the conventional materials. Consequently, MSCs that are isolated, stored, and grown using the xeno-free materials including the serum-free medium (MesenCult-XF™), TC protector™, and recombinant trypsin (TrypLESelect™) might be applicable for regenerative medicine of cartilage.     

Effects of mechanical stimulation on the proliferation of bone marrow-derived human mesenchymal stem cells

To support and enhance thein vitro growth and activity of mesenchymal stem cells (MSCs), the cell culture medium may be supplemented with various proteins and factors to mimic the physiological environment in which the cells optimally proliferate and differentiate. In this study, the effects of mechanical factors on cellular metabolic responses were investigated experimentally using a bioreactor. The effects of various chemical factors, such as growth factors, cytokines, and hormones, were also investigated. Based on previous reports demonstrating the important roles of mechanical factors in the growth and activity of MSCs, we sought to evaluate the effects of mechanical stimuli on the proliferation of bone marrow-derived MSCs using a cell training bioreactor that imposed cyclic mechanical stretch, with parameters of 240 min/day, 0.03 Hz, and 5–15% strain. The application of cyclic stretch (5–15% strain) to the MSCs enhanced their proliferation during the early stage (3 days), but not the late stage (14 days), of batch culture. Mechanical stretch did not increase the release of lactate dehydrogenase (LDH) from the MSCs during culture. Appropriate levels of mechanical stretch (5–10% strain) increased collagen synthesis, but did not alter MSC surface antigen expression. It is thought that the appropriate level of mechanical stretch was able to serve as a potent positive modulator of MSC proliferation during the initial stages of culture.     

AcSDKP抑制体外培养条件下人骨髓间充质干细胞的增殖

N-乙酰基-丝氨酰-天冬氨酰-赖氨酰-脯氨酸(N-acetyl-seryl-aspartyl-lysyl-proline,AcSDKP)是一种具有生理调控活性的四肽因子,对造血干/祖细胞增殖具有抑制作用。本研究采用集落形成实验、甲基偶氮唑盐(MTT)比色法、细胞分裂指数测定等方法,考察了AcSDKP对体外培养的人骨髓间充质干细胞(mesenchymal stem cell,MSC)增殖的影响。结果显示,在AcSDKP浓度为1×10-12mol／L-1×10-9mol／L的培养体系中,人骨髓MSC集落生成率和大小、活力细胞数和分裂指数均降低,最大效应浓度为1×10-11mol／L。以上实验结果表明,在体外培养条件下,一定浓度的AcSDKP对人骨髓MSC 的增殖具有抑制作用。     

Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells

Bone marrow mesenchymal stem cells (MSCs) are capable of differentiating into a variety of cell types such as vascular smooth muscle cells (SMCs). In this study, we investigated influence of cyclic stretch on proliferation of hMSCs for different loading conditions, alignment of actin filaments, and consequent differentiation to SMCs. Isolated cells from bone marrow were exposed to cyclic stretch utilizing a customized device. Cell proliferation was examined by MTT assay, alignment of actin fibers by a designed image processing code, and cell differentiation by fluorescence staining. Results indicated promoted proliferation of hMSCs by cyclic strain, enhanced by elevated strain amplitude and number of cycles. Such loading regulated smooth muscle α-actin, and reoriented actin fibers. Cyclic stretch led to differentiation of hMSCs to SMCs without addition of growth factor. It was concluded that applying appropriate loading treatment on hMSCs could enhance proliferation capability, and produce functional SMCs for engineered tissues.     

Effects of human full-length amelogenin on the proliferation of human mesenchymal stem cells derived from bone marrow

Amelogenins are enamel matrix proteins that play a crucial role in enamel formation. Recent studies have revealed that amelogenins also have cell signaling properties. Although amelogenins had been described as specific products of ameloblasts, recent research has demonstrated their expression in bone marrow stromal cells. In this study, we examined the effect of recombinant human full-length amelogenin (rh174) on the proliferation of human mesenchymal stem cells (MSCs) derived from bone marrow and characterized the associated changes in intracellular signaling pathways. MSCs were treated with rh174 ranging in dose from 0 to 1,000 ng/ml. Cell proliferative activity was analyzed by bromodeoxyuridine (BrdU) immunoassay. The expression of lysosomal-associated membrane protein 1 (LAMP1), a possible amelogenin receptor, in MSCs was analyzed. Anti-LAMP1 antibody was used to block the binding of rh174 to LAMP1. The MAPK-ERK pathway was examined by Cellular Activation of Signaling ELISA (CASE) kit and western blot analysis. A specific MAPK inhibitor, U0126, was used to block ERK activity. It was shown that rh174 increased the proliferation of MSCs and MAPK-ERK activity. The MSC proliferation and MAPK-ERK activity enhanced by rh174 were reduced by the addition of anti-LAMP1 antibody. Additionally, the increased proliferation of MSCs induced by rh174 was inhibited in the presence of U0126. In conclusion, it is demonstrated that rh174 increases the proliferation of MSCs by interaction with LAMP1 through the MAPK-ERK signaling pathway, indicating the possibility of MSC application to tissue regeneration in the orofacial region.     

Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells

The presence within bone marrow of a population of mesenchymal stem cells (MSCs) able to differentiate into a number of different mesenchymal tissues, including bone and cartilage, was first suggested by Friedenstein nearly 40 years ago. Since then MSCs have been demonstrated in a variety of fetal and adult tissues, including bone marrow, fetal blood and liver, cord blood, amniotic fluid and, in some circumstances, in adult peripheral blood. MSCs from all of these sources can be extensively expanded in vitro and when cultured under specific permissive conditions retain their ability to differentiate into multiple lineages including bone, cartilage, fat, muscle, nerve, glial and stromal cells. There has been great interest in these cells both because of their value as a model for studying the molecular basis of differentiation and because of their therapeutic potential for tissue repair and immune modulation. However, MSCs are a rare population in these tissues. Here we tried to identify cells with MSC-like potency in human placenta. We isolated adherent cells from trypsin-digested term placentas and examined these cells for morphology, surface markers, and differentiation potential and found that they expressed several stem cell markers. They also showed endothelial and neurogenic differentiation potentials under appropriate conditions. We suggest that placenta-derived cells have multilineage differentiation potential similar to MSCs in terms of morphology and cell-surface antigen expression. The placenta may prove to be a useful source of MSCs.     

Effects of malondialdehyde on growth and proliferation of human bone marrow mesenchymal stem cells in vitro

Malondialdehyde(MDA)is a well known inducer of carbonyl stress in a variety of human cells,however,its effects on human bone marrow mesenchymal stem cells(hMSCs)have not been documented.In this study,the effects of MDA concentration on the growth rate and proliferation of hMSCs in vitro were assessed.Under high concentrations of MDA,the cell count was decreased and the population doubling time(PDT)was lengthened.Flow cytometry(FCM)demonstrated that MDA triggered cells to undergo apoptosis.in parallel with the findings in MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]assay which showed that it can also impair cellular viability.Surprisingly,FCM also determined that the percentage of hMSCs in G2/M- and S-phases also increased in a dose-dependent manner with respect to MDA concentration.These results strongly suggest that even though hMSCs were severely impaired by high concentrations of MDA,they were still able to send signals that resulted in accelerated cellular proliferation process.This study provided important insights on how carbonyl stress affects cell cycle and proliferation of hMSCs.     

Tissue-engineered bone formation with cryopreserved human bone marrow mesenchymal stem cells

Bone marrow mesenchymal stem cells (MSCs) have become the main cell source for bone tissue engineering. It has been reported that cryopreserved human MSCs can maintain their potential for proliferation and osteogenic differentiation in vitro. There are, however, no reports on osteogenesis with cryopreserved human MSCs in vivo. The aim of this study was to determine whether cryopreservation had an effect on the proliferation capability and osteogenic differentiation of human MSCs on scaffolds in vitro and in vivo. MSCs were isolated from human bone marrow, cultured in vitro until passage 2, and then frozen and stored at −196 °C in liquid nitrogen with 10% Me₂SO as cryoprotectant for 24 h. The cryopreserved MSCs were then thawed rapidly, seeded onto partially demineralized bone matrix (pDBM) scaffolds and cultured in osteogenic media containing 10 mM sodium β-glycerophosphate, 50 μM l-ascorbic acid, and 10 nM dexamethasone. Non-cryopreserved MSCs seeded onto the pDBM scaffolds were used as control groups. Scanning electronic microscopy (SEM) observation, DNA content assays, and measurements of alkaline phosphatase (ALP) activity and osteocalcin (OCN) content were applied, and the results showed that the proliferation potential and osteogenic differentiation of MSCs on pDBM in vitro were not affected by cryopreservation. After 2 weeks of subculture, the MSCs/pDBM composites were subcutaneously implanted into the athymic mice. The constructs were harvested at 4 and 8 weeks postimplantation, and histological examination showed tissue-engineered bone formation in the pDBM pores in both groups. Based on these results, it can be concluded that cryopreservation allows human MSCs to be available for potential therapeutic use to tissue-engineer bone.     

Cell proliferation of human bone marrow mesenchymal stem cells on biodegradable microcarriers enhances in vitro differentiation potential

Objectives: For reasons of provision of highly‐specific surface area and three‐dimensional culture, microcarrier culture (MC) has garnered great interest for its potential to expand anchorage‐dependent stem cells. This study utilizes MC for in vitro expansion of human bone marrow mesenchymal stem cells (BMMSCs) and analyses its effects on BMMSC proliferation and differentiation. Materials and methods: Effects of semi‐continuous MC compared to control plate culture (PC) and serial bead‐to‐bead transfer MC (MC bead‐T) on human BMMSCs were investigated. Cell population growth kinetics, cell phenotypes and differentiation potential of cells were assayed. Results: Maximum cell density and overall fold increase in cell population growth were similar between PCs and MCs with similar starting conditions, but lag period of BMMSC growth differed substantially between the two; moreover, MC cells exhibited reduced granularity and higher CXCR4 expression. Differentiation of BMMSCs into osteogenic and adipogenic lineages was enhanced after 3 days in MC. However, MC bead‐T resulted in changes in cell granularity and lower osteogenic and adipogenic differentiation potential. Conclusions: In comparison to PC, MC supported expansion of BMMSCs in an up‐scalable three‐dimensional culture system using a semi‐continuous process, increasing potential for stem cell homing ability and osteogenic and adipogenic differentiation.     

Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability of BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0mMSr(2+)) under osteogenic or adipogenic medium for 1 and 2weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPARγ2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPARγ in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.     

Analysis of neuron-like differentiation of human bone marrow mesenchymal stem cells

The objective of the study was to evaluate differentiation of human bone marrow mesenchymal stem cells into true or pseudo neurons after treating with chemical induction medium in vitro. The morphological changes were assessed using interference contrast microscopy. Immunocytochemistry and Western blotting were performed using neuronal markers. Further evaluation was conducted with proteomic profiling, DNA microarray analysis and the whole-cell patch clamp test. After three hours of treatment with chemical induction medium, nearly three-fourths of the hMSCs changed to cells with a neuronal phenotype. The results of immunocytochemistry and Western blotting showed a high expression of neuronal markers in these cells at 3 h which decreased at 24 h. The proteomics analysis showed no change of proteins related to neuronal differentiation. DNA microarray showed downregulation of neuron related genes. The patch clamp test was unable to demonstrate any similarity to true neurons. Our findings suggest that neuron-like cells derived from chemical induction of hMSCs are not the genuine neurons as they resemble true neurons phenotypically but are different in genotypic and electrophysiological characteristics.     

Effects of malondialdehyde on growth and proliferation of human bone marrow mesenchymal stem cells in vitro

Malondialdehyde (MDA) is a well known inducer of carbonyl stress in a variety of human cells, however, its effects on human bone marrow mesenchymal stem cells (hMSCs) have not been documented. In this study, the effects of MDA concentration on the growth rate and proliferation of hMSCs in vitro were assessed. Under high concentrations of MDA, the cell count was decreased and the population doubling time (PDT) was lengthened. Flow cytometry (FCM) demonstrated that MDA triggered cells to undergo apoptosis, in parallel with the findings in MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay which showed that it can also impair cellular viability. Surprisingly, FCM also determined that the percentage of hMSCs in G₂/M-and S-phases also increased in a dose-dependent manner with respect to MDA concentration. These results strongly suggest that even though hMSCs were severely impaired by high concentrations of MDA, they were still able to send signals that resulted in accelerated cellular proliferation process. This study provided important insights on how carbonyl stress affects cell cycle and proliferation of hMSCs. __________ Translated from Journal of Natural Science of Hunan Normal University, 2005, 28 (2) [译自: 湖南师范大学自然科学报, 2005,28(2)]     

Effect of pulsed electromagnetic field on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells

Pulsed electromagnetic fields (PEMFs) have been used clinically to slow down osteoporosis and accelerate the healing of bone fractures for many years. The aim of this study is to investigate the effect of PEMFs on the proliferation and differentiation potential of human bone marrow mesenchymal stem cells (BMMSC). PEMF stimulus was administered to BMMSCs for 8 h per day during culture period. The PEMF applied consisted of 4.5 ms bursts repeating at 15 Hz, and each burst contained 20 pulses. Results showed that about 59% and 40% more viable BMMSC cells were obtained in the PEMF‐exposed cultures at 24 h after plating for the seeding density of 1000 and 3000 cells/cm², respectively. Although, based on the kinetic analysis, the growth rates of BMMSC during the exponential growth phase were not significantly affected, 20–60% higher cell densities were achieved during the exponentially expanding stage. Many newly divided cells appeared from 12 to 16 h after the PEMF treatment as revealed by the cell cycle analysis. These results suggest that PEMF exposure could enhance the BMMSC cell proliferation during the exponential phase and it possibly resulted from the shortening of the lag phase. In addition, according to the cytochemical and immunofluorescence analysis performed, the PEMF‐exposed BMMSC showed multi‐lineage differentiation potential similar to the control group. Bioelectromagnetics 30:251–260, 2009. © 2009 Wiley‐Liss, Inc.     

Non-hematopoietic human bone marrow contains long-lasting, pluripotential mesenchymal stem cells

Mesenchymal stem cells (MSC) are considered as potential agents for reconstructive and gene-targeting therapies since they differentiate into various cell-lineages, exhibit an extended survival once injected into a host, and can easily be transfected with engineered DNA. MSC are essentially isolated from hematopoietic bone marrow (BM), a process that is rather invasive and may raise ethical concerns. In an attempt to find an alternative source, we evaluated whether non-hematopoietic (nh)BM recovered from femoral heads of patients undergoing hip arthroplasty contained MSC. Ex vivo, 99% of nhBM cells were CD45(+) leukocytes. After culture, leukocytes were replaced by a homogeneous layer of adherent CD45(-) CD14(-) CD34(-) CD11b(-) CD90(+) HLA-ABC(+) cells. Culture doubling time (mean = 4 days, range 1.6-6.7 days) was not correlated with patient age (27-81 years, n = 16). Amplified cultures supported long-term hematopoiesis, and could be differentiated in vitro into adipocytes and chondrocytes. Moreover, a small fraction of nhBM cells spontaneously expressed MyoD1 and formed myotubes, suggesting that myogenic differentiation also occurred. nhBM contained clonogenic cells whose frequency (1/13,000), doubling time (2.1 days), and maximal amplification (up to 10(6)-fold) were not age-related. All 14 clones analyzed (from five patients, ages 27-78 years) differentiated into at least one mesenchymal lineage, and 66% were bipotential (n = 8/12), or tripotential (n = 2/3). In conclusion, nhBM contains pluripotential mesenchymal progenitors which are similar to hematopoietic BM-derived MSC, and whose biological functions are not altered by aging. Furthermore, if MSC-based therapies hold their promises, nhBM may become the source of choice for responding to the increasing demand for MSC.     

Gene delivery to human bone marrow mesenchymal stem cells by microporation

Electroporation has been considered one of the most efficient non-viral based methods to deliver genes regardless of frequently observed high cell mortality. In this study we used a microporation technique to optimise the delivery of plasmid DNA encoding green fluorescence protein (GFP) to human bone marrow mesenchymal stem cells (BM-MSC). Using resuspension buffer (RB) and as low as 1.5 × 10⁵ cells and 1 μg of DNA, we achieved 40% of cells expressing the transgene, with cell recovery and cell viabilities of 85% and 90%, respectively. An increase in DNA amount did not significantly increase the number of transfected cells but clearly reduced cell recovery. A face-centered composite design was used to unveil the conditions giving rise to optimal plasmid delivery efficiencies when using a sucrose based microporation buffer (SBB). The BM-MSC proliferation kinetics were mainly affected by the presence of plasmid and not due to the microporation process itself although no effect was observed on their immunophenotypic characteristics and differentiative potential. Based on the data shown herein microporation demonstrated to be a reliable and efficient method to genetically modify hard-to-transfect cells giving rise to the highest levels of cell survival reported so far along with superior gene delivery efficiencies.