Secretion of oestrogen from murine-induced pluripotent stem cells co-cultured with ovarian granulosa cells in vitro

POF (premature ovarian failure) is a distressing condition that is a common cause of infertility. No effective treatment is available to overcome the loss of fertility. A method to derive oestrogen from miPSCs (mouse-induced pluripotent stem cells) was explored as a potential treatment for POF. In this study, C57BL/6 female mice were injected with PMSG (pregnant mare's serum gonadotropin) to obtain ovarian GCs (granulosa cells) and then co-cultured with miPSCs. The morphological changes in the miPSCs co-cultured with GCs were observed by light microscopy. The expression of FSHR (follicle-stimulating hormone receptor) was detected by immunocytochemistry and flow cytometry. Radioimmunoassay was used to analyse the level of E2 (oestradiol) in culture supernatants. The results showed that the proportion of GCs expressing FSHR in GCs was over 90%. The E2 concentration of the culture supernatant of the GC group was 62.4 pg/ml on day 1 and decreased in a time-dependent manner. The opposite situation was observed in the miPSCs-GC co-cultured group with an E2 concentration of 87.9 pg/ml on day 1 that increased in a time-dependent manner to reach a concentration of 328.4 pg/ml on day 7. The data indicate that GC-like cells were effectively induced from miPSCs through indirect cell-to-cell contact. Our method provides a novel in vitro system to study miPSC differentiation, particularly the interactions between miPSCs and GCs. The ultimate goal of this approach would be to provide a treatment for POF in the future.     

Isolation of neural crest-derived stem cells from rat embryonic mandibular processes

BACKGROUND INFORMATION: Substantial evidence indicates the existence of NCSCs (neural crest-derived stem cells) in embryonic mandibular processes; however, they have not been fully investigated or isolated. The aim of the present study was to isolate stem cells from mandibular process during embryonic development by MACS (magnetic-activated cell sorting). The findings show that the cells are multipotent and self-renewing. RESULTS: LNGFR (low-affinity nerve-growth-factor receptor)+ cells were isolated from rat embryonic mandibular processes by MACS. The cells were grown in clonal culture by limiting dilution to assess their developmental potential. Clone analysis indicated that, first, LNGFR+ cells are multipotent, being able to generate at least neurons and Schwann cells, similar to peripheral neural crest stem cells. Secondly, multipotent LNGFR+ cells generate multipotent progenies, indicating that they are capable of self-renewal and therefore are stem cells. Thirdly, manipulation of the medium supplementation alters the fate of the isolated LNGFR+ cells. CONCLUSIONS: These results indicate that LNGFR antibodies label NCSCs with high specificity and purity, and suggest that positive selection using these antibodies may become the method of choice for obtaining multipotent cells from rat embryonic mandibular processes for tissue engineering or regenerative therapeutic use.     

Expression of GD2 and GD3 gangliosides in human embryonic neural stem cells

NSCs (neural stem cells) are undifferentiated neural cells endowed with a high potential for proliferation and a capacity for self-renewal with retention of multipotency to differentiate into neurons and glial cells. It has been recently reported that GD3, a b-series ganglioside, is a marker molecule for identifying and isolating mouse NSCs. However, the expression of gangliosides in human NSCs is largely unknown. In the present study, we analysed the expression of gangliosides, GD2 and GD3, in human NSCs that were isolated from human brains at gestational week 17 in the form of neurospheres, which are floating clonal aggregates formed by NSCs in vitro. Employing immunocytochemistry, we found that human NSCs were strongly reactive to anti-GD2 antibody and relatively weakly reactive to anti-GD3 antibody. Treatment of these cells with an organic solvent such as 100% methanol, which selectively removes glycolipids from plasma membrane, abolished the immunoreactivity with those antibodies, indicating that the reactivity was due to GD2 and GD3, but not to GD2-/GD3-like glycoproteins or proteoglycans. The immunoreactivity of human NSCs to antibody against SSEA-1 (stage-specific embryonic antigen-1), a well-known carbohydrate antigen of NSCs, was not decreased by the treatment with 100% methanol, indicating that SSEA-1 is mainly carried by glycoproteins and/or proteoglycans in human NSCs. Our study suggests that GD2 and GD3 can be marker gangliosides for identifying human NSCs.     

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell's replication activity and the donor's age factor, and to assess the stem cells as a new source for tissue engineering, hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds ＜20 years old, 21-40years old, 41-60 years old and ＞61 years old groups). The protein markers (CD29, CD34, CD44, CD45,CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell,and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro.The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula "TD = t log2/logNt - logN0 "was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the ＜20 years old group was lower than that of the ＞61 years old group (statistical analysis of variance (ANOVA), P=-0.002, P＜0.05). These findings suggested that a higher level of hADAS cells replication activity was found in the younger donators, and they represent novel and valuable seed cells for studies of tissue engineering.     

Biological character of human adipose-derived adult stem cells and influence of donor age on cell replication in culture

To investigate the biological character of human adipose-derived adult stem cells (hADAS cells) when cultured in vitro and the relationship between hADAS cell’s replication activity and the donor’s age factor, and to assess the stem cells as a new source for tissue engineering. hADAS cells are isolated from human adipose tissue of different age groups (from adolescents to olds: <20 years old, 21–40 years old, 41–60 years old and >61 years old groups). The protein markers (CD29, CD34, CD44, CD45, CD49d, HLA-DR, CD106) of hADAS cells were detected by flow cytometry (FCM) to identify the stem cell, and the cell cycle was examined for P20 hADAS cells to evaluate the safety of the subculture in vitro. The generative activity of hADAS cells in different age groups was also examined by MTT method. The formula “TD = t log2/logNt − logN0” was used to get the time doubling (TD) of the cells. The results showed that the cells kept heredity stabilization by chromosome analysis for at least 20 passages. The TD of these cells increased progressively by ageing, and the TD of the <20 years old group was lower than that of the >61 years old group (statistical analysis of variance (ANOVA), P=0.002, P<0.05). These findings suggested that a higher level of hADAS cells replication activity was found in the younger donators, and they represent novel and valuable seed cells for studies of tissue engineering.     

Effect and mechanism of mGluR6 on the biological function of rat embryonic neural stem cells

Here, we investigated the effects and molecular mechanisms of metabotropic glutamate receptor 6 (mGluR6) on rat embryonic neural stem cells (NSCs). Overexpression of mGluR6 significantly promoted the proliferation of NSCs and increased the diameter of neutrospheres after treatment for 24 h, 48 h and 72 h. Overexpression of mGluR6 promoted G1 to S phase transition, with significantly decreased cell ratio in G1/G0 phase but significantly increased cell ratio in S phase. Additionally, mGluR6 overexpression for 48 h decreased the early and late apoptosis significantly. Moreover, overexpression of mGluR6 significantly increased the expression of p-ERK1/2, Cyclin D1 and CDK2, while the expression of p-p38 was significantly decreased. On the contrary, these effects of mGluR6 overexpression were reversed by mGluR6 knockdown. In conclusion, mGluR6 promotes the proliferation of NSCs by activation of ERK1/2-Cyclin D1/CDK2 signaling pathway and inhibits the apoptosis of NSCs by blockage of the p38 MAPK signaling pathway.     

Mouse 3T3 fibroblasts under the influence of fibroblasts isolated from stroma of human basal cell carcinoma acquire properties of multipotent stem cells

Background information. Multipotent mesenchymal stem cells can participate in the formation of a microenvironment stimulating the aggressive behaviour of cancer cells. Moreover, cells exhibiting pluripotent ESC (embryonic stem cell) markers (Nanog and Oct4) have been observed in many tumours. Here, we investigate the role of cancer‐associated fibroblasts in the formation of stem cell supporting properties of tumour stroma. We test the influence of fibroblasts isolated from basal cell carcinoma on mouse 3T3 fibroblasts, focusing on the expression of stem cell markers and plasticity in vitro by means of microarrays, qRT‐PCR (quantitative real‐time PCR) and immunohistochemistry. Results. We demonstrate the biological activity of the cancer stromal fibroblasts by influencing the 3T3 fibroblasts to express markers such as Oct4, Nanog and Sox2 and to show differentiation potential similar to mesenchymal stem cells. The role of growth factors such as IGF2 (insulin‐like growth factor 2), FGF7 (fibroblast growth factor 7), LEP (leptin), NGF (nerve growth factor) and TGFβ (transforming growth factor β), produced by the stromal fibroblasts, is established to participate in their bioactivity. Uninduced 3T3 do not express the stem cell markers and show minimal differentiation potential. Conclusions. Our observations indicate the pro‐stem cell activity of cancer‐associated fibroblasts and underline the role of epithelial—mesenchymal interaction in tumour biology.     

Fivefold increase in derivation rates of mouse embryonic stem cells after supplementation of the media with multiple factors

The objective of this study was to determine the ability of multiple-factor supplementation to augment derivation of mouse embryonic stem (mES) cells. Three factors, leukemia inhibitory factor (LIF), Parke-Davis 98059 (PD98059), and 6-bromoindirubin-3′-oxime (BIO), were added as supplements (individually or in a combination of all three) at two consecutive stages of culture; that is, from the start of blastocyst culture to the outgrowth stage, and from putting disaggregated outgrowth into culture medium to generation of primary mES colonies, respectively. The main outcome measure was the percentage of derivable mES cell lines, based on the number of blastocysts initially cultured. Three experiments demonstrated the following: (1) For the addition of individual single factor, only LIF yielded mES cell lines (6.2%), whereas a combination of all three factors resulted in the greatest number of mES cell lines (31.3%). (2) The advantages of a combination of multiple factors (LIF + PD98059 + BIO) were manifested only when they were used during the first stage of the culture and not during the second stage (31.6% vs. 6.2%, respectively). (3) The quality of the inner cell mass (ICM) outgrowth obtained from first-stage culture was studied. After alkaline phosphatase and Oct-4 staining, which documented pluripotency of the embryonic stem cells, outgrowths cultured in multiple factors (LIF + PD98059 + BIO) stained much stronger and in higher proportions than did those obtained after supplementation only with LIF (80% vs. 30%, respectively).     

Efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging(1-3). Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells(1-3). Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration(1,4-7). However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity(7-10). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic(11-13). To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules(14) (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders(1, 14). And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons(1, 14, 15). However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.     

Proliferation and differentiation potential of human adipose-derived mesenchymal stem cells isolated from elderly patients with osteoporotic fractures

Aging has less effect on adipose-derived mesenchymal stem cells (ADSCs) than on bone marrow-derived mesenchymal stem cells (BMSCs), but whether the fact holds true in stem cells from elderly patients with osteoporotic fractures is unknown. In this study, ADSCs and BMSCs of the same donor were harvested and divided into two age groups. Group A consisted of 14 young patients (36.4 ± 11.8 years old), and group B consisted of eight elderly patients (71.4 ± 3.6 years old) with osteoporotic fractures. We found that the doubling time of ADSCs from both age groups was maintained below 70 hrs, while that of BMSCs increased significantly with the number of passage. When ADSCs and BMSCs from the same patient were compared, there was a significant increase in the doubling time of BMSCs in each individual from passages 3 to 6. On osteogenic induction, the level of matrix mineralization of ADSCs from group B was comparable to that of ADSCs from group A, whereas BMSCs from group B produced least amount of mineral deposits and had a lower expression level of osteogenic genes. The p21 gene expression and senescence-associated β-galactosidase activity were lower in ADSCs compared to BMSCs, which may be partly responsible for the greater proliferation and differentiation potential of ADSCs. It is concluded that the proliferation and osteogenic differentiation of ADSCs were less affected by age and multiple passage than BMSCs, suggesting that ADSCs may become a potentially effective therapeutic option for cell-based therapy, especially in elderly patients with osteoporosis.     

Efficient derivation of human cardiac precursors and cardiomyocytes from pluripotent human embryonic stem cells with small molecule induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.     

The role of the N-methyl-d-aspartate receptor in the proliferation of adult hippocampal neural stem and precursor cells

New neurons are continuously generated from resident pools of neural stem and precursor cells(NSPCs)in the adult brain.There are multiple pathways through which adult neurogenesis is regulated,and here we review the role of the N-methyl-D-aspartate receptor(NMDAR)in regulating the proliferation of NSPCs in the adult hippocampus.Hippocampal-dependent learning tasks,enriched environments,running,and activity-dependent synaptic plasticity,all potently up-regulate hippocampal NSPC proliferation.We first consider the requirement of the NMDAR in activity-dependent synaptic plasticity,and the role the induction of synaptic plasticity has in regulating NSPCs and newborn neurons.We address how specific NMDAR agonists and antagonists modulate proliferation,both in vivo and in vitro,and then review the evidence supporting the hypothesis that NMDARs are present on NSPCs.We believe it is important to understand the mechanisms underlying the activation of adult neurogenesis,given the potential that endogenous stem cell populations have for repopulating the hippocampus with functional new neurons.In conditions such as age-related memory decline,neurodegeneration and psychiatric disease,mature neurons are lost or become defective;as such,stimulating adult neurogenesis may provide a therapeutic strategy to overcome these conditions.     

Abscisic acid switches cell division modes of asymmetric cell division and symmetric cell division in stem cells of protonemal filaments in the moss Physcomitrium patens

Multicellular organisms regulate cell numbers and cell fate by using asymmetric cell division (ACD) and symmetric cell division (SCD) during their development and to adapt to unfavorable environmental conditions. A stem cell self-renews and generates differentiated cells. In plants, various types of cells are produced by ACD or SCD; however, the molecular mechanisms of ACD or SCD and the cell division mode switch are largely unknown. The moss Physcomitrium (Physcomitrella) patens is a suitable model to study plant stem cells due to its simple anatomy. Here, we report the cell division mode switch induced by abscisic acid (ABA) in P. patens. ABA is synthesized in response to abiotic stresses and induces round-shape cells, called brood cells, from cylindrical protonemal cells. Although two daughter cells with distinct sizes were produced by ACD in a protonemal stem cell on ABA-free media, the sizes of two daughter cells became similar with ABA treatment. Actin microfilaments were spatially localized on the apices of apical stem cells in protonemata on ABA-free media, but the polar accumulation was lost under the condition of ABA treatment. Moreover, ABA treatment conferred an identical cell fate to the daughter cells in terms of cell division activity. Collectively, the results indicate ABA may suppress the ACD characteristics but evoke SCD in cells. We also noticed that ABA-induced brood cells not only self-renewed but regenerated protonemal cells when ABA was removed from the media, suggesting that brood cells are novel stem cells that are induced by environmental signals in P. patens.     

Ovarian Stem Cells (OSCs) from the Cryopreserved Ovarian Cortex: A Potential for Neo-Oogenesis in Women with Cancer-Treatment Related Infertility: A Case Report and a Review of Literature

Cancer treatment related infertility (CTRI) affects more than one third of young women undergoing anti-cancer protocols, inducing a premature exhaustion of the ovarian reserve. In addition to ovarian suppression by GnRHa, oocyte and cortex cryopreservation has gained interest in patients with estrogen-sensitive tumors for whom the hormonal burst to prompt the multiple follicular growth could provide a further pro-life tumor pulsing. On the other hand, cortex reimplantation implies a few drawbacks due to the unknown consistency of the follicles to be reimplanted or the risk of reintroducing malignant cells. The capability of ovarian stem cells (OCSs) from fresh ovarian cortex fragments to differentiate in vitro to mature oocytes provides a tool to overcome these drawbacks. In fact, since ovarian cortex sampling and cryopreservation is practicable before gonadotoxic treatments, the recruitment of OSCs from defrosted fragments could provide a novel opportunity to verify their suitability to be expanded in vitro as oocyte like cells (OLCs). Here, we describe in very preliminary experiments the consistency of an OSC population from a single cryopreserved ovarian cortex after thawing as well as both their viability and their suitability to be further explored in their property to differentiate in OLCs, thus reinforcing interest in stemness studies in the treatment of female CTRI.     

Roles for Kisspeptin in proliferation and differentiation of spermatogonial cells isolated from mice offspring when the cells are cocultured with somatic cells

Kisspeptin (Kp) expression in testis has caused most of the recent research surveying its functional role in this organ. This peptide influences spermatogenesis and sperm capacitation, so it is considered as a regulator of reproduction. Kp roles exert through hypothalamic/pituitary/gonadal axis. We aimed to evaluate direct roles for Kp on proliferation and differentiation of spermatogonial cells (SCs) when the cells are cocultured with somatic cells. Somatic cells and SCs were isolated from adult azoospermic and newborn mice and then enriched using a differential attachment technique. After the evaluation of identity and colonization for SCs, the cells were cocultured with somatic cells, and three doses of Kp (10⁻⁸-10⁻⁶ M) was assessed on proliferation (through evaluation of MVH and ID4 markers) and differentiation (via evaluation of c-Kit and SCP₃, TP_1, TP₂, and, Prm₁ markers) of the coculture system. Investigations were continued for four succeeding weeks. At the end of each level of testosterone in the culture media was also evaluated. We found positive influence from Kp on proliferative and differentiative markers in SCs cocultured with somatic cells. These effects were dose-dependent. There was no effect for Kp on testosterone level. From our findings, we simply conclude that Kp as a neuropeptide for influencing central part of reproductive axis could also positively affect peripheral processes related to spermatogenesis without having an effect on steroidogenesis.     

Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells

The optimization of a purely negative depletion, enrichment process for circulating tumor cells (CTCs) in the peripheral blood of head and neck cancer patients is presented. The enrichment process uses a red cell lysis step followed by immunomagnetic labeling, and subsequent depletion, of CD45 positive cells. A number of relevant variables are quantified, or attempted to be quantified, which control the performance of the enrichment process. Six different immunomagnetic labeling combinations were evaluated as well as the significant difference in performance with respect to the blood source: buffy coats purchased from the Red Cross, fresh, peripheral blood from normal donors, and fresh peripheral blood from human cancer patients. After optimization, the process is able to reduce the number of normal blood cells in a cancer patient's blood from 4.05 × 10⁹ to 8.04 × 10³ cells/mL and still recover, on average, 2.32 CTC per mL of blood. For all of the cancer patient blood samples tested in which CTC were detected (20 out of 26 patients) the average recovery of CTCs was 21.7 per mL of blood, with a range of 282 to 0.53 CTC. Since the initial number of CTC in a patient's blood is unknown, and most probably varies from patient to patient, the recovery of the CTC is unknown. However, spiking studies of a cancer cell line into normal blood, and subsequent enrichment using the optimized protocol indicated an average recovery of approximately 83%. Unlike a majority of other published studies, this study focused on quantifying as many factors as possible to facilitate both the optimization of the process as well as provide information for current and future performance comparisons. The authors are not aware any other reported study which has achieved the performance reported here (a 5.66 log₁₀) in a purely negative enrichment mode of operation. Such a mode of operation of an enrichment process provides significant flexibility in that it has no bias with respect to what attributes define a CTC; thereby allowing the researcher or clinician to use any maker they choose to define whether the final, enrich product contains CTCs or other cell type relevant to the specific question (i.e., does the CTC have predominately epithelial or mesenchymal characteristics?). Biotechnol. Bioeng. 2009;102: 521–534. © 2008 Wiley Periodicals, Inc.