Proliferation and differentiation of spermatogonial stem cells

Spermatogonial stem cells (A(s) spermatogonia) are single cells that either renew themselves or produce A(pr) (paired) spermatogonia predestined to differentiate. In turn, the A(pr) divide into chains of A(al) (aligned) spermatogonia that also divide. The ratio between self-renewal and differentiation of the stem cells is regulated by glial cell line-derived neurotrophic factor produced by Sertoli cells, while the receptors are expressed in stem cells. A(s), A(pr) and A(al) spermatogonia proliferate during part of the epithelial cycle forming many A(al) spermatogonia. During epithelial stage VIII, almost all A(al) spermatogonia, few A(pr) and very few A(s) spermatogonia differentiate into A1 spermatogonia. A number of molecules are involved in this differentiation step including the stem cell factor-c-kit system, the Dazl RNA binding protein, cyclin D(2) and retinoic acid. There is no fine regulation of the density of spermatogonial stem cells and consequently, in some areas, many A1 and, in other areas, few A1 spermatogonia are formed. An equal density of spermatocytes is then obtained by the apoptosis of A2, A3 or A4 spermatogonia to remove the surplus cells. The Bcl-2 family members Bax and Bcl-x(L) are involved in this density regulation. Several mechanisms are available to cope with major or minor shortages in germ cell production. After severe cell loss, stem cell renewal is preferred above differentiation and the period of proliferation of A(s), A(pr) and A(al) spermatogonia is extended. Minor shortages are dealt with, at least in part, by less apoptosis among A2-A4 spermatogonia.     

Chlorogenic acid promotes osteoblastogenesis in human adipose tissue-derived mesenchymal stem cells

High-fat diet (HFD)-induced obesity is associated with oxidative stress. The purpose of this study was to examine the antioxidant effect of Phaeodactylum tricornutum extract in mice with diet-induced obesity. Four-week-old C57BL/6J mice were fed a normal diet or HFD with and without 0.7% P. tricornutum lipid extract corresponding to 0.2% fucoxanthin for 8 weeks. P. tricornutum significantly decreased body weight and epidydimal white adipose tissue in mice fed the HFD. Serum triglyceride, glucose, insulin, and leptin levels, as well as homeostasis model assessment for insulin resistance (HOMA-IR) values, were significantly lower in the P. tricornutum group than in the HFD group. P. tricornutum significantly decreased thiobarbituric acid reactive substances (TBARS) and increased glutathione and the activities of superoxide dismutase, catalase, and glutathione peroxidase in the liver compared with the HFD group. Thus, P. tricornutum could exert antiobesity and antioxidant effects in mice fed a HFD.     

High inoculation cell density could accelerate the differentiation of human bone marrow mesenchymal stem cells to chondrocyte cells

The effects of the density of human mesenchymal stem cells (MSCs) on their differentiation to chondrocytes in a differentiation medium supplemented with dexamethasone, TGFbeta3, and IGF-1 were investigated for the regenerative therapy of cartilage. The increase in the initial density of MSCs from 0.05 x 10(4) to 0.9 x 10(4) cells/cm(2) accelerated the increase in the expression level of aggrecan mRNA during the differentiation culture for 7 d. The conditioned medium harvested at 7 d from the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2) accelerated the initial increase in the expression level for 3 d in the differentiation culture with an initial MSC density of 0.3 x 10(4) cells/cm(2), whereas the conditioned medium harvested at 7 d in the differentiation culture with an initial MSC density of 0.05 x 10(4) cells/cm(2) did not. The differentiation culture after 14 d with an initial MSC concentration of 0.3 x 10(4) cells/cm(2) showed an expression level 1.7-fold that in the case of the culture with an initial MSC concentration of 0.05 x 10(4) cells/cm(2). Thus, a high MSC inoculum density might be appropriate for the rapid differentiation of MSCs to chondrocytes.     

Effect of ascorbic acid on bone marrow-derived mesenchymal stem cell proliferation and differentiation

Mesenchymal stem cells (MSCs) derived from bone marrow are an important tool in tissue engineering and cell-based therapies because of their multipotent capacity. Majority of studies on MSCs have investigated the roles of growth factors, cytokines, and hormones. Antioxidants such as ascorbic acid can be used to expand MSCs while preserving their differentiation ability. Moreover, ascorbic acid can also stimulate MSC proliferation without reciprocal loss of phenotype and differentiation potency. In this study, we evaluated the effects of ascorbic acid on the proliferation, differentiation, extracellular matrix (ECM) secretion of MSCs. The MSCs were cultured in media containing various concentrations (0-500 microM) of L-ascorbate-2-phosphate (Asc-2-P) for 2 weeks, following which they were differentiated into adipocytes and osteoblasts. Ascorbic acid stimulated ECM secretion (collagen and glycosaminoglycan) and cell proliferation. Moreover, the phenotypes of the experimental groups as well as the differentiation potential of MSCs remained unchanged. The apparent absence of decreased cell density or morphologic change is consistent with the toxicity observed with 5-250 microM concentrations of Asc-2-P. The results demonstrate that MSC proliferation or differentiation depends on ascorbic acid concentration.     

Myricetin inhibits adipogenesis in human adipose tissue-derived mesenchymal stem cells

Myricetin is a major flavonoid found in various foods that has antioxidant, anti-inflammatory, and anticancer effects. Although the functional effects of myricetin in various cell types are well characterized, it is not known whether myricetin has an effect on stem cell differentiation. In this study, we demonstrate that myricetin inhibits adipogenesis in human adipose tissue-derived mesenchymal stem cells, as indicated by decreased accumulation of intracellular lipid droplets. The mRNA levels of CCAATenhancer-binding proteins (C/EBP)-α, peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase, fatty acid binding protein (aP2), and adiponectin decreased significantly following treatment with 30 μM myricetin. C/EBP-α expression was inhibited from the beginning of differentiation in response to the myricetin treatment. PPAR-α was significantly inhibited beginning at day 9. These results suggest a novel effect of myricetin on adipocyte differentiation in human adipose tissue-derived mesenchymal stem cells and the possibility that myricetin might affect the differentiation of other types of stem cells.     

Glucose-stimulated insulin secretion of various mesenchymal stem cells after insulin-producing cell differentiation

Mesenchymal stem cells (MSCs) are capable of crossing germinative layer borders and are obtainable in high numbers via in vitro cultures. Therefore, many researchers have searched for diverse sources of MSCs. Recently the generation of glucose-responsive insulin-producing cells (IPCs) from MSCs has shown immense potential for the treatment of type 1 diabetes mellitus (T1DM) due to a lack of pancreas donors. In this study, we compared the growth potency of four kinds of MSCs derived from bone marrow, Wharton's jelly, adipose tissue, and the periosteum. In addition, in vitro differentiation of these MSCs into IPCs was also investigated. After 2weeks of IPCs differentiation, we compared the expression of the insulin gene and protein using RT-qPCR and immunofluorescence staining. Only IPCs derived from periosteum-derived progenitor cells (PDPCs) showed a response to glucose concentration. Glucose stimulated insulin secretion was conclusive evidence of the potential functionality of IPCs. Therefore, PDPCs are a promising alternative stem cell source for IPCs differentiation.     

Influence of fetal calf serum on differentiation of mesenchymal stem cells to chondrocytes during expansion

Mesenchymal stem cells (MSCs) should be expanded in vitro while maintaining their multilineage potential before differentiation to one mesenchymal lineage for application to regeneration therapy. The effect of fetal calf serum (FCS) on undesirable differentiation during subcultivations for the expansion was investigated. The expression level of the aggrecan gene, which is a marker of chondrogenic differentiation, gradually and markedly increased during the subcultivations of MSCs with the addition of 10% FCS and without additional cytokines. The percentage of cells positive for CD90 and CD166, which are markers of MSCs, decreased, and the percentage of large polygonal cells and the average cell adhesion area increased during the expansion. There was a marked difference in the increase in the aggrecan expression level between the two expansion cultures employing different FCS lots, although their proliferation rates were almost the same. The decrease in FCS concentration resulted in a higher percentage of CD90(+)CD166(+) cells, a lower percentage of large polygonal cells, and a lower level of aggrecan expression. Consequently, FCS components could stimulate MSC differentiation to chondrocytes and a lower concentration could decrease this differentiation.     

Combination of cytomegalovirus enhancer with human cellular promoters for gene-induced chondrogenesis of human bone marrow mesenchymal stem cells

High-expression plasmid vectors for human mesenchymal stem cells (MSCs) were constructed by combination of cytomegalovirus immediate-early enhancer with cellular promoters. MSCs transfected with the vector showed higher transgene production of a cytokine, which increased the differentiation level to chondrocytes.     

Shrinkage-free preparation of scaffold-free cartilage-like disk-shaped cell sheet using human bone marrow mesenchymal stem cells

Aiming for the clinical application of cartilage regeneration, a culture method for mesenchymal stem cells (MSCs) derived from human bone marrow to obtain scaffold-free cartilage-like disk-shaped sheet of uniform sizes without the shrinkage was investigated. A disk-shaped cell sheet having the same diameter as that of the membrane without the shrinkage was formed after the cultivation of MSCs (18.6 × 10(5)cells/well) for 3 weeks in a cell culture insert (CCI) containing a flat membrane whose porosity was 12%, while 6.2 and 31.0 × 10(5)MSCs/well, respectively, resulted in the shrinkage of the aggregate and the hole formation in the center part of the sheet. Cell aggregates shrunk also in a 96-well plate and CCIs having lower porosity. The disk-shaped cell sheet showed the comparable thickness (1.2mm) and sulfated glycosaminoglycan (sGAG) density to those of the pellet formed in a pellet culture. The gene expression levels of aggrecan and type II collagen in the disk-shaped cell sheet were not lower than those in the pellet. In conclusion, the usage of CCI having 12% porosity and 18.6 × 10(5)MSCs/well could avoid the shrinkage from the formation of the scaffold-free cartilage-like disk-shaped cell sheet.     

Porcine uterus contains a population of mesenchymal stem cells

The uterus has a remarkable ability of cycling remodeling throughout the reproductive life of the female. Recent findings in the human and mouse indicate that adult stem/progenitor cells may play a prominent role in the maintenance of uterine endometrial and myometrial homeostasis. We aimed to characterize the prospective stem/progenitor cells in the porcine uterus and establish a new model for uterine stem cell research. In this study, we demonstrated that cells isolated from porcine uterus have capacity for in vitro differentiation into adipogenic and osteogenic lineages and express the mesenchymal stem cell (MSC) markers CD29, CD44, CD144, CD105, and CD140b as revealed by RT-PCR. Moreover, we showed that some cells isolated from the porcine uterus when cultured at low density produce large clones with an efficiency of 0.035%. Simultaneously, they were negative for hematopoietic stem cell markers such as CD34 and CD45. Low expression of nestin, which is specific for neural stem cells and various progenitor cells, was also detected. We conclude that the porcine uterus contains a small population of undifferentiated cells with MSC-like properties similar to human and mouse uteri.     

Isolation, characterization and differentiation of mesenchymal stem cells from amniotic fluid, umbilical cord blood and Wharton's jelly in the horse

Mesenchymal stem cells (MSCs) have been derived from multiple sources of the horse including umbilical cord blood (UCB) and amnion. This work aimed to identify and characterize stem cells from equine amniotic fluid (AF), CB and Wharton's Jelly (WJ). Samples were obtained from 13 mares at labour. AF and CB cells were isolated by centrifugation, while WJ was prepared by incubating with an enzymatic solution for 2 h. All cell lines were cultured in DMEM/TCM199 plus fetal bovine serum. Fibroblast-like cells were observed in 7/10 (70%) AF, 6/8 (75%) CB and 8/12 (66.7%) WJ samples. Statistically significant differences were found between cell-doubling times (DTs): cells isolated from WJ expanded more rapidly (2.0±0.6 days) than those isolated from CB (2.6±1.3 days) and AF (2.3±1.0 days) (P<0.05). Positive von Kossa and Alizarin Red S staining confirmed osteogenesis. Alcian Blue staining of matrix glycosaminoglycans illustrated chondrogenesis and positive Oil Red O lipid droplets staining suggested adipogenesis. All cell lines isolated were positive for CD90, CD44, CD105; and negative for CD34, CD14 and CD45. These findings suggest that equine MSCs from AF, UCB and WJ appeared to be a readily obtainable and highly proliferative cell lines from a uninvasive source that may represent a good model system for stem cell biology and cellular therapy applications in horses. However, to assess their use as an allogenic cell source, further studies are needed for evaluating the expression of markers related to cell immunogenicity.     

Transition of mechanical property of porous alginate scaffold with cells during culture period

The rupture forces of porous alginate scaffolds seeded with hepatocytes or fibroblast-like cells increased during 3 d of culture and then decreased. The phenomenon was independent of the number of viable cells within the scaffolds, but dependent on protein adsorption to the alginate as well as a reduction in the degree of crosslinks of the calcium-alginate gel.     

Germ cells from mouse and human embryonic stem cells

Mammalian gametes are derived from a founder population of primordial germ cells (PGCs) that are determined early in embryogenesis and set aside for unique development. Understanding the mechanisms of PGC determination and differentiation is important for elucidating causes of infertility and how endocrine disrupting chemicals may potentially increase susceptibility to congenital reproductive abnormalities and conditions such as testicular cancer in adulthood (testicular dysgenesis syndrome). Primordial germ cells are closely related to embryonic stem cells (ESCs) and embryonic germ (EG) cells and comparisons between these cell types are providing new information about pluripotency and epigenetic processes. Murine ESCs can differentiate to PGCs, gametes and even blastocysts - recently live mouse pups were born from sperm generated from mESCs. Although investigations are still preliminary, human embryonic stem cells (hESCs) apparently display a similar developmental capacity to generate PGCs and immature gametes. Exactly how such gamete-like cells are generated during stem cell culture remains unclear especially as in vitro conditions are ill-defined. The findings are discussed in relation to the mechanisms of human PGC and gamete development and the biotechnology of hESCs and hEG cells.     

Comparison of efficiency of terminal differentiation of oligodendrocytes from induced pluripotent stem cells versus embryonic stem cells in vitro

Oligodendrocytes are the myelinating cells of the central nervous system (CNS), and defects in these cells can result in the loss of CNS functions. Although oligodendrocyte progenitor cells transplantation therapy is an effective cure for such symptoms, there is no readily available source of these cells. Recent studies have described the generation of induced pluripotent stem cells (iPS cells) from somatic cells, leading to anticipation of this technique as a novel therapeutic tool in regenerative medicine. In this study, we evaluated the ability of iPS cells derived from mouse embryonic fibroblasts to differentiate into oligodendrocytes and compared this with the differential ability of mouse embryonic stem cells (ES cells). Experiments using an in vitro oligodendrocyte differentiation protocol that was optimized to ES cells demonstrated that 2.3% of iPS cells differentiated into O4⁺ oligodendrocytes compared with 24.0% of ES cells. However, the rate of induction of A2B5⁺ oligodendrocyte precursor cell (OPC) was similar for both iPS-derived cells and ES-derived cells (14.1% and 12.6%, respectively). These findings suggest that some intracellular factors in iPS cells inhibit the terminal differentiation of oligodendrocytes from the OPC stage.