Characterization and expression analysis of mesenchymal stem cells from human bone marrow and adipose tissue.

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering.     

Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.     

Effect of isolation methodology on stem cell properties and multilineage differentiation potential of human dental pulp stem cells

Dental pulp stem cells (DPSCs) are an attractive alternative mesenchymal stem cell (MSC) source because of their isolation simplicity compared with the more invasive methods associated with harvesting other MSC sources. However, the isolation method to be favored for obtaining DPSC cultures remains under discussion. This study compares the stem cell properties and multilineage differentiation potential of DPSCs obtained by the two most widely adapted isolation procedures. DPSCs were isolated either by enzymatic digestion of the pulp tissue (DPSC-EZ) or by the explant method (DPSC-OG), while keeping the culture media constant throughout all experiments and in both isolation methods. Assessment of the stem cell properties of DPSC-EZ and DPSC-OG showed no significant differences between the two groups with regard to proliferation rate and colony formation. Phenotype analysis indicated that DPSC-EZ and DPSC-OG were positive for CD29, CD44, CD90, CD105, CD117 and CD146 expression without any significant differences. The multilineage differentiation potential of both stem cell types was confirmed by using standard immuno(histo/cyto)chemical staining together with an in-depth ultrastructural analysis by means of transmission electron microscopy. Our results indicate that both DPSC-EZ and DPSC-OG could be successfully differentiated into adipogenic, chrondrogenic and osteogenic cell types, although the adipogenic differentiation of both stem cell populations was incomplete. The data suggest that both the enzymatic digestion and outgrowth method can be applied to obtain a suitable autologous DPSC resource for tissue replacement therapies of both bone and cartilage.     

Immortalized mouse dental papilla mesenchymal cells preserve odontoblastic phenotype and respond to bone morphogenetic protein 2

Odontogenesis is the result of the reciprocal interactions between epithelial–mesenchymal cells leading to terminally differentiated odontoblasts. This process from dental papilla mesenchymal cells to odontoblasts is regulated by a complex signaling pathway. When isolated from the developing tooth germs, odontoblasts quickly lose their potential to maintain the odontoblast-specific phenotype. Therefore, generation of an odontoblast-like cell line would be a good surrogate model for studying the dental mesenchymal cell differentiation into odontoblasts and the molecular events of dentin formation. In this study, immortalized dental papilla mesenchymal cell lines were generated from the first mouse mandibular molars at postnatal day 3 using pSV40. These transformed cells were characterized by RT-PCR, immunohistochemistry, Western blot, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iMDP-3, displayed a high proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers and demonstrated the ability to differentiate and form mineralized nodules. Furthermore, iMDP-3 cells had high transfection efficiency as well as were inducible and responded to BMP2 stimulation. We conclude that the establishment of the stable murine dental papilla mesenchymal cell line might be used for studying the mechanisms of dental cell differentiation and dentin formation.     

A comprehensive study on optimization of proliferation and differentiation potency of bone marrow derived mesenchymal stem cells under prolonged culture condition

Bone marrow derived stem cells (BMSC) have paved way to clinical approaches for its utilization in a variety of diseases due to its ease of isolation combined with its multilineage differentiation capacity. However, the applicability of BMSC is not successful due to the lesser number of nucleated cells obtained from large samples. Hence, culture expansion of BMSC is a prerequisite, as high numbers of stem cells are needed to meet the standards of clinical advancement. There are attempts on optimizing culture condition for large scale production of BMSC. It was believed that, prolonged culture of BMSC is difficult since they tend to lose their characteristics and differentiation potential. Hence, our study aims to determine whether BMSCs could retain its proliferative and differentiation capacity in prolonged in vitro culture by a comparative study on extensive culturing of BMSC with the following four media, DMEM LG (DMEM-Low Glucose), DMEM KO (DMEM-Knock Out), Alpha MEM (Alpha Minimal Essential Medium), DMEM F 12. We found that two samples among the three cultured tend to lose their property in long term culturing. Besides, we also found that DMEM LG and Alpha MEM were the optimal media for in vitro culturing of BMSC. Overall, it was concluded that BMSC can be cultured until passage 15 without losing its characteristics. However, its potency beyond passage 15 has to be further elucidated for utilization of the ex vivo expanded BMSC for subsequent cellular therapies.     

Multilineage differentiation potential of equine blood-derived fibroblast-like cells

Tissue engineering (TE) has emerged as a promising new therapy for the treatment of damaged tissues and organs. Adult stem cells are considered as an attractive candidate cell type for cell-based TE. Mesenchymal stem cells (MSC) have been isolated from a variety of tissues and tested for differentiation into different cell lineages. While clinical trials still await the use of human MSC, horse tendon injuries are already being treated with autologous bone marrow-derived MSC. Given that the bone marrow is not an optimal source for MSC due to the painful and risk-containing sampling procedure, isolation of stem cells from peripheral blood would bring an attractive alternative. Adherent fibroblast-like cells have been previously isolated from equine peripheral blood. However, their responses to the differentiation conditions, established for human bone marrow MSC, were insufficient to fully confirm their multilineage potential. In this study, differentiation conditions were optimized to better evaluate the multilineage capacities of equine peripheral blood-derived fibroblast-like cells (ePB-FLC) into adipogenic, osteogenic, and chondrogenic pathways. Adipogenic differentiation using rabbit serum resulted in a high number of large-size lipid droplets three days upon induction. Cells' expression of alkaline phosphatase and calcium deposition upon osteogenic induction confirmed their osteogenic differentiation capacities. Moreover, an increase of dexamethasone concentration resulted in faster osteogenic differentiation and matrix mineralization. Finally, induction of chondrogenesis in pellet cultures resulted in an increase in cartilage-specific gene expression, namely collagen II and aggrecan, followed by protein deposition after a longer induction period. This study therefore demonstrates that ePB-FLC have the potential to differentiate into adipogenic, osteogenic, and chondrogenic mesenchymal lineages. The presence of cells with confirmed multilineage capacities in peripheral blood has important clinical implications for cell-based TE therapies in horses.     

Scanning electron microscopy preparation protocol for differentiated stem cells

The lack of an established protocol for scanning electron microscopy (SEM) studies on stem cells differentiating into adipogenic lineage led us to develop a protocol for the preparation of differentiated adult bone marrow-derived mesenchymal stem cells (BMSC) for SEM. This protocol describes the procedure to maintain and preserve the structural organization of cellular components following differentiation, for morphological and physical characterization. The fixation of the differentiated cells was followed by dehydration using methanol, and vacuum desiccation before microscopy. The use of longer chain alcohols as dehydrating agents was avoided in our method to reduce the dissolution of lipid deposits in cells, thus allowing the maintenance of their structural integrity. The time period for the processing of samples was reduced by avoiding the osmium tetroxide postfixation and critical point drying. Thus, this protocol helps in determining the potential, fate, and degree of stem cell differentiation. This may be useful for SEM analysis of differentiated cells, especially those grown on various scaffolds.     

Comparison between isolation protocols highlights intrinsic variability of human umbilical cord mesenchymal cells

Mesenchymal stem cells (MSCs) though multipotent exhibit limited lifespan in vitro, with progressive reduction in capacity for self-renewal leading to irreversible arrest of cell division, which limits their use for therapeutic purposes. Human umbilical cord wall MSCs are easy to process and proliferate rapidly in culture, but variability of individual samples and impact upon in vitro expansion and aging processes is unknown. We compared isolation protocols to determine which one yields the highest number of viable cells with the best proliferation capacity. Three different protocols were tested: two were enzymatic procedures and one explant method. Isolated cells were evaluated in terms of proliferation, differentiation capacity, and phenotype. All samples were processed using one or more protocols. After passage 2 adherent cells displayed standard phenotypic and differentiation characteristics of MSCs, but our results show that isolating cells directly from Wharton’s jelly is more advantageous. Cells obtained from explants presented similar characteristics to those from enzymatic protocols, but always reached proliferation arrest earlier, irrespective of initial population doubling times. From the same sample, cells obtained with enzymatic protocol ii reached later passages while exhibiting shorter doubling times in culture than cells from other protocols, that is, took longer to reach senescence. More important, each individual MSC sample exhibited different population doubling rates and reached senescence at different passages, irrespective of protocol. Thus, even when in strict conformity with procedures and quality control, each cord sample shows a unique behavior, a finding that should be taken into account when planning for therapeutic approaches.     

Biological characterization and pluripotent identification of ovine amniotic fluid stem cells

Mesenchymal stem cells derived from amniotic fluid have become one of the most potential stem cell source for cell-based therapy for the reason they can be harvested at low cost and without ethical problems. Here, we obtained amniotic fluid stem cells (AFSCs) from ovine amniotic fluid and studied the expansion capacity, cell markers expression, karyotype, and multilineage differentiation ability. In our work, AFSCs were subcultured to passage 62. The cell markers, CD29, CD44, CD73 and OCT4 which analyzed by RT-PCR were positive; CD44, CD73, CD90, CD105, NANOG, OCT4 analyzed by immunofluorescence and flow cytometry were also positive. The growth curves of different passages were all typically sigmoidal. The different passages cells took on a normal karyotype. In addition, AFSCs were successfully induced to differentiate into adipocytes, osteoblasts and chondrocytes. The results suggested that the AFSCs isolated from ovine maintained normal biological characteristics and their multilineage differentiation potential provides many potential applications in cell-based therapies and tissue engineering.     

Retention of multilineage differentiation potential of mesenchymal cells during proliferation in response to FGF

Mesenchymal stem cells (MSC) that can differentiate to various connective tissue cells may be useful for autologous cell transplantation to defects of bone, cartilage, and tendon, if MSC can be expanded in vitro. However, a short life span of MSC and a reduction in their differentiation potential in culture have limited their clinical application. The purpose of this study is to identify a growth factor(s) involved in self-renewal of MSC and the maintenance of their multilineage differentiation potential. Fibroblast growth factor-2 (FGF-2) markedly increased the growth rate and the life span of rabbit, canine, and human bone marrow MSC in monolayer cultures. This effect of FGF-2 was more prominent in low-density cultures than in high-density cultures. In addition, all MSC expanded in vitro with FGF-2, but not without FGF-2, differentiated to chondrocytes in pellet cultures. The FGF+ MSC also retained the osteogenic and adipogenic potential throughout many mitotic divisions. These findings suggest that FGFs play a crucial role in self-renewal of MSC.     

Isolation and characterization of equine amniotic fluid-derived multipotent stem cells

Background aimsAmniotic fluid (AF) is a well-known source of stem cells. However, there have been no reports regarding equine AF stem cells. We have isolated equine AF-derived multipotent stem cells (MSC) (eAF-MSC) and show that these cells exhibit self-renewal ability and multilineage differentiation.MethodsAF was obtained from thoroughbred mares and mononuclear cells (MNC) were isolated by Ficoll–Paque density gradient. We measured the cumulative population doubling level (CPDL) and characterized the immunophenotype by flow cytometry. To investigate differentiation ability, a trilineage differentiation assay was conducted.ResultseAF-MSC could be isolated and the proliferation level was high. eAF-MSC presented typical MSC phenotypic markers, as determined by flow cytometry. Moreover, eAF-MSC showed a trilineage differentiation capability.ConclusionsEquine AF is a good source of MSC. Furthermore, eAF-MSC may be useful as a cell therapy application for horses.     

Human mesenchymal stem cells from the umbilical cord matrix: Successful isolation and ex vivo expansion using serum-/xeno-free culture media

Mesenchymal stem cells (MSC) could potentially be applied in therapeutic settings due to their multilineage differentiation ability, immunomodulatory properties, as well as their trophic activity. The umbilical cord matrix (UCM) represents a promising source of MSC for biomedical applications. The number of cells isloated per umbilical cord (UC) unit is limited and ex vivo expansion is imperative in order to reach clinically meaningful cell numbers. The limitations of poorly defined reagents (e.g. fetal bovine serum, which is commonly used as a supplement for human MSC expansion) make the use of serum-/xeno-free conditions mandatory. We demonstrated the feasibility of isolating UCM-MSC by plastic adherence using serum-/xeno-free culture medium following enzymatic digestion of UCs, with a 100% success rate. 2.6 ± 0.21 × 10⁵ cells were isolated per UC unit, of which 1.9 ± 0.21 × 10⁵ were MSC-like cells expressing CD73, CD90, and CD105. When compared to adult sources (bone marrow-derived MSC and adipose-derived stem/stromal cells), UCM-MSC displayed a similar immunophenotype and similar multilineage differentiation ability, while demonstrating a higher expansion potential (average fold increase of 7.4 for serum-containing culture medium and 11.0 for xeno-free culture medium (P3-P6)). The isolation and expansion of UCM-MSC under defined serum-/xeno-free conditions contributes to safer and more effective MSC cellular products, boosting the usefulness of MSC in cellular therapy and tissue engineering.     

Isolation of embryonic stem-like cells from equine blastocysts and their differentiation in vitro

Embryonic stem (ES) cells are pluripotent cells with the potential capacity to generate any type of cell. We describe here the isolation of pluripotent ES-like cells from equine blastocysts that have been frozen and thawed. Our two lines of ES-like cells (E-1 and E-2) appear to maintain a normal diploid karyotype indefinitely in culture in vitro and to express markers that are characteristic of ES cells from mice, namely, alkaline phosphatase, stage-specific embryonic antigen-1, STAT-3 and Oct 4. After culture of equine ES-like cells in vitro for more than 17 passages, some ES-like cells differentiated to neural precursor cells in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor and platelet-derived growth factor. We also developed a protocol that resulted in the differentiation of ES-like cells in vitro to hematopoietic and endothelial cell lineages in response to bFGF, stem cell factor and oncostatin M. Our observations set the stage for future developments that may allow the use of equine ES-like cells for the treatment of neurological and hematopoietic disorders.     

Effect of Donor Age on the Proportion of Mesenchymal Stem Cells Derived from Anterior Cruciate Ligaments

The characteristics of anterior cruciate ligament (ACL)-derived mesenchymal stem cells (MSCs), such as proportion and multilineage potential, can be affected by donor age. However, the qualitative and quantitative features of ACL MSCs isolated from younger and older individuals have not yet been compared directly. This study assessed the phenotypic and functional differences in ACL-MSCs isolated from younger and older donors and evaluated the correlation between ACL-MSC proportion and donor age. Torn ACL remnants were harvested from 36 patients undergoing ACL reconstruction (young: 29.67 ± 10.92 years) and 33 undergoing TKA (old: 67.96 ± 5.22 years) and the proportion of their MSCs were measured. The mean proportion of MSCs was slightly higher in older ACL samples of the TKA group than of the younger ACL reconstruction group (19.69 ± 8.57% vs. 15.33 ± 7.49%, p = 0.024), but the proportions of MSCs at passages 1 and 2 were similar. MSCs from both groups possessed comparable multilineage potentiality, as they could be differentiated into adipocytes, osteocytes, and chondrocytes at similar level. No significant correlations were observed between patient age and MSC proportions at passages 0–2 or between age and MSC proportion in both the ACL reconstruction and TKA groups. Multiple linear regression analysis found no significant predictor of MSC proportion including donor age for each passage. Microarray analysis identified several genes that were differentially regulated in ACL-MSCs from old TKA patients compared to young ACL reconstruction patients. Genes of interest encode components of the extracellular matrix (ECM) and may thus play a crucial role in modulating tissue homeostasis, remodeling, and repair in response to damage or disease. In conclusion, the proportion of freshly isolated ACL-MSC was higher in elderly TKA patients than in younger patients with ACL tears, but their phenotypic and multilineage potential were comparable.     

Undifferentiated mouse mesenchymal stem cells spontaneously express neural and stem cell markers Oct-4 and Rex-1

BACKGROUND: Previous adult stem cells studies have provided evidence that BM mesenchymal stem cells (MSC) exhibit multilineage differentiation capacity. These properties of MSC prompted us to explore the neural potential of MSC with a view to their use for the treatment of demyelinating disorders, such as multiple sclerosis. Indeed, issues such as the identification of a subset of stem cells that is neurally fated, methods of expansion and optimal stage of differentiation for transplantation remain poorly understood. METHODS: In order to isolate mouse (m) MSC from BM, we used and compared the classic plastic-adhesion method and one depleting technique, the magnetic-activated cell sorting technique. RESULTS: We established and optimized culture conditions so that mMSC could be expanded for more than 360 days and 50 passages. We also demonstrated that undifferentiated mMSC express the neural markers nestin, MAP2, A2B5, GFAP, MBP, CNPase, GalC, O1 under standard culture conditions before transplantation. The pluripotent stem cell marker Oct-4 and the embryonic stem cell marker Rex-1 are spontaneously expressed by untreated mMSC. The lineage-negative mMSC (CD5- CD11b- Ly-6G- Ter119- CD45R- c-kit/CD117-) overexpressed Oct-4, O1 and A2B5 in the first days of culture compared with the non-sorted MSC. Finally, we identified a distinct subpopulation of mMSC that is primed towards a neural fate, namely Sca-1+/nestin+ mMSC. DISCUSSION: These results should facilitate the optimal timing of harvesting a neurally fated subpopulation of mMSC for transplantation into animal models of human brain diseases.     

Neural differentiation ability of mesenchymal stromal cells from bone marrow and adipose tissue: a comparative study

Background aimsThe characteristics, such as morphologic and phenotypic characteristics and neural transdifferentiation ability, of mesenchymal stromal cells (MSC) derived from different origins have yet to be reported for cases isolated from the same individual.MethodsThe proliferation capacity, secretion ability of neurotrophins (NT) and neural differentiation ability in rat MSC isolated from bone marrow (BMSC) and adipose tissue (ADSC) were compared from the same animal.ResultsThe ADSC had a significantly higher proliferation capacity than BMSC according to cell cycle and cumulative population doubling analyses. The proportion of cells expressing neural markers was greater in differentiated ADSC than in differentiated BMSC. Furthermore, the single neurosphere derived from ADSC showed stronger expansion and differentiation abilities than that derived from BMSC. The findings from Western blot lent further support to the immunocytochemical data. The mRNA and protein levels of nerve growth factor (NGF) and brain-derived growth factor (BDNF) expressed in ADSC were significantly higher than those in BMSC at different stages before and following induction.ConclusionsOur study suggests that the proliferation ability of ADSC is superior to that of BMSC. Furthermore, differentiated ADSC expressed higher percentages of neural markers. As one possible alternative source of BMSC, ADSC may have wide potential for treating central nervous system (CNS) diseases.     

Chemical engineering of mesenchymal stem cells to induce a cell rolling response

Covalently conjugated sialyl Lewis X (SLeX) on the mesenchymal stem cell (MSC) surface through a biotin-streptavidin bridge imparts leukocyte-like rolling characteristics without altering the cell phenotype and the multilineage differentiation potential. We demonstrate that the conjugation of SLeX on the MSC surface is stable, versatile, and induces a robust rolling response on P-selectin coated substrates. These results indicate the potential to increase the targeting efficiency of any cell type to specific tissue.