Cardiac differentiation at an initial low density of human-induced pluripotent stem cells

A high density of human-induced pluripotent stem cells (hiPSCs) improves the efficiency of cardiac differentiation, suggesting the existence of indispensable cell-cell interaction signals. The complexity of interactions among cells at high density hinders the understanding of the roles of cell signals. In this study, we determined the minimum cell density that can initiate differentiation to facilitate cell-cell interaction studies. First, we co-induced cardiac differentiation in the presence of the glycogen synthase kinase-3β inhibitor CHIR99021 and activin A at various cell densities. At an initial low density, cells died within a few days in RPMI-based medium. We then investigated the culture conditions required to maintain cell viability. We used a basal medium excluding important components for the maintenance of hiPSC pluripotency, including activin A, basic fibroblast growth factor, and insulin. Supplementation of the basal medium with Rho-associated protein kinase inhibitor and insulin improved cell viability. Interestingly, addition of basic fibroblast growth factor enabled the expression of cardiac markers at the mRNA level but not the protein level. After further modification of the culture conditions, 10% of the cells expressed the cardiac troponin T protein, which is associated with cell contraction. The novel protocol for cardiac differentiation at an initial low cell density can also be used to evaluate high cell density conditions. The findings will facilitate the identification of cell signals required for cardiomyocyte formation.     

A primary response with an initial negative wave

In the anterosuperior and posterosuperior portions of the ectosylvian gyrus of dogs small foci were found in which clicks evoked responses with parameters close to those of primary responses (PRs), but with an initial negative wave. An analysis of PRs with an initial wave of different polarity was carried out by studying the reproducibility of the response during an increase in the frequency of stimulation and during the action of various drugs. PRs with an initial negative ("negative" focus) and positive (auditory area AI) waves were found to be reproduced when the frequency of stimulation was 20–30 Hz, whereas the negative phase of the classical PR disappeared when the stimulation frequency reached 10–15 Hz. The polarity of the response in these foci was unchanged after injection of a lethal dose of nembutal, but the negative phase of the classical PR disappeared during moderately deep anesthesia. Strychnine, on the other hand, considerably increased the amplitude of the negative wave in the positive-negative complex, but the initial negative potential was only very slightly and temporarily increased, and it was lost in the subsequent strychnine spike. GABA inhibited both the PR with initial negative wave and also the negative phase of the classical PR. The results suggest that PRs with different polarities of their initial wave differ in origin. The results of experiments with GABA indicate that PRs with an initial negative wave arise through excitation of apical dendrites.A. A. Bogomolets' Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 5, pp. 488–496, September–October, 1970.     

Multilineage differentiation of human MSC after in utero transplantation

Prenatal transplantation of stem cells is an exciting frontier for the treatment of many congenital diseases. The fetus may be an ideal recipient for stem cells, as it is immunologically immature and has rapidly proliferating cellular compartments that may support the engraftment of transplanted cells. Mesenchymal stem cells (MSC), given their ability to differentiate among multiple lineages, could potentially be used to treat diseases such as osteogenesis imperfecta, muscular dystrophy, and a variety of others that can be diagnosed in utero. We have shown, using a human-sheep in utero xenotransplantation model, that human MSC have the ability to engraft, differentiate into many tissue types, and survive for over 1 year in fetal lamb recipients. This observation warrants further studies of the behavior of MSC following systemic or site-directed transplantation.     

Interaction of MSC with tumor cells

Tumor development and tumor progression is not only determined by the corresponding tumor cells but also by the tumor microenvironment. This includes an orchestrated network of interacting cell types (e.g. immune cells, endothelial cells, fibroblasts, and mesenchymal stroma/stem cells (MSC)) via the extracellular matrix and soluble factors such as cytokines, chemokines, growth factors and various metabolites. Cell populations of the tumor microenvironment can interact directly and indirectly with cancer cells by mutually altering properties and functions of the involved partners. Particularly, mesenchymal stroma/stem cells (MSC) play an important role during carcinogenesis exhibiting different types of intercellular communication. Accordingly, this work focusses on diverse mechanisms of interaction between MSC and cancer cells. Moreover, some functional changes and consequences for both cell types are summarized which can eventually result in the establishment of a carcinoma stem cell niche (CSCN) or the generation of new tumor cell populations by MSC-tumor cell fusion.     

Transcriptional profiling of initial differentiation events in human embryonic stem cells

Currently, there are no differentiation strategies for human embryonic stem cells (hESCs) that efficiently produce one specific cell type, possibly because of lack of understanding of the genes that control signaling events prior to overt differentiation. sed HepG2 cell conditioned medium (MEDII), which induces early differentiation in mouse ES cells while retaining pluripotent markers, to query gene expression in hESCs. Treatment of adherent hESCs with 50% MEDII medium effected differentiation to a cell type with gene expression similar to primitive streak stage cells of mouse embryos. MEDII treatment up-regulates TDGF1 (Cripto), a gene essential for anterior-posterior axis and mesoderm formation in mouse embryos and a key component of the TGFB1/NODAL signaling pathway. LEFTYA, an antagonist of NODAL/TDGF1 signaling expressed in anterior visceral endoderm, is down-regulated with MEDII treatment, as is FST, an inhibitor of mesoderm induction via the related INHBE1 pathway. In summary, the TGFB1/NODAL pathway is important for primitive-streak and mesoderm formation and in using MEDII, we present a means for generating an in vitro cell population that maintains pluripotent gene expression (POU5F1, NANOG) and SSEA-4 markers while regulating genes in the TGFB1/NODAL pathway, which may lead to more uniform formation of mesoderm in vitro.     

Conventional, naive CD4+ T cells provide an initial source of IL-4 during Th2 differentiation

IL-4 is known to promote the differentiation of CD4+ T cells into IL-4-secreting Th2 cells. However, the cellular source of the early burst of IL-4 that drives Th2 responses in vivo has not been conclusively identified. Mice deficient for the IL-4 receptor alpha-chain (IL-4Ralpha-/-) retain the capacity to secrete IL-4 and can be used to identify those cell types that produce IL-4 without a requirement for prior IL-4-mediated stimulation. To address whether naive, conventional CD4+ T cells may act as initial producers of IL-4 in Ag-specific responses, we crossed the BALB/c IL-4Ralpha-/-mice to DO11.10/scid TCR transgenic mice. Lymph node cells from wild-type and IL-4Ralpha-/- DO11.10/scid mice secreted approximately 50 pg of IL-4 per10(6) cells within 48 h after peptide stimulation. This small amount of IL-4 was sufficient to cause the differentiation of wild-type CD4+ T cells into Th2 cells, particularly if IFN-gamma and IL-12 were neutralized during the priming cultures. CD4+ cells from the IL-4Ralpha-/- mice gave rise to a minor proportion (approximately 2%) of IL-4-producing cells upon stimulation in the presence of anti-IFN-gamma and anti-IL-12. These data show that conventional, naive CD4+ T cells may be considered as initial sources of IL-4 and, in the absence of IFN-gamma and IL-12, this IL-4 can induce Th2 polarization.     

Telomerase inhibition promotes an initial step of cell differentiation of primate embryonic stem cell

Embryonic stem (ES) cell is well known as a totipotent cell, which is derived from a blastcyst and has potential to differentiate into every kind of somatic cell. ES cell bears self-renewal characteristic as well as differentiation potential. ES cell bears telomerase activity to avoid telomere shortening, which is a characteristic of differentiated somatic cells. As the differentiation of ES cells proceeds, their telomerase activity is losing. However, it has not been convinced whether suppression of the telomerase activity promotes progression of ES cell differentiation. The effect of telomerase inhibitor on the differentiation potential of marmoset ES cell was assessed, counting cells expressing embryonic markers (alkaline phosphatase and TPA-1-60) under existence of a telomerase inhibitor. Telomerase inhibitor showed a promotional effect for the marmoset ES cell differentiation. This result suggests that exogenous inhibition of telomerase activity leads to induction of an early differentiation of primate ES cell.     

Promotion of hematopoietic differentiation from mouse induced pluripotent stem cells by transient HoxB4 transduction

Ectopic expression of HoxB4 in embryonic stem (ES) cells leads to an efficient production of hematopoietic cells, including hematopoietic stem/progenitor cells. Previous studies have utilized a constitutive HoxB4 expression system or tetracycline-regulated HoxB4 expression system to induce hematopoietic cells from ES cells. However, these methods cannot be applied therapeutically due to the risk of transgenes being integrated into the host genome. Here, we report the promotion of hematopoietic differentiation from mouse ES cells and induced pluripotent stem (iPS) cells by transient HoxB4 expression using an adenovirus (Ad) vector. Ad vector could mediate efficient HoxB4 expression in ES cell-derived embryoid bodies (ES-EBs) and iPS-EBs, and its expression was decreased during cultivation, showing that Ad vector transduction was transient. A colony-forming assay revealed that the number of hematopoietic progenitor cells with colony-forming potential in HoxB4-transduced cells was significantly increased in comparison with that in non-transduced cells or LacZ-transduced cells. HoxB4-transduced cells also showed more efficient generation of CD41-, CD45-, or Sca-1-positive cells than control cells. These results indicate that transient, but not constitutive, HoxB4 expression is sufficient to augment the hematopoietic differentiation of ES and iPS cells, and that our method would be useful for clinical applications, such as cell transplantation therapy.     

Degenerate wave and capacitive coupling increase human MSC invasion and proliferation while reducing cytotoxicity in an in vitro wound healing model

Non-unions pose complications in fracture management that can be treated using electrical stimulation (ES). Bone marrow mesenchymal stem cells (BMMSCs) are essential in fracture healing; however, the effect of different clinical ES waveforms on BMMSCs cellular activities remains unknown. We compared the effects of direct current (DC), capacitive coupling (CC), pulsed electromagnetic field (PEMF) and degenerate wave (DW) on cellular activities including cytotoxicity, proliferation, cell-kinetics and apoptosis by stimulating human-BMMSCs 3 hours a day, up to 5 days. In addition, migration and invasion were assessed using fluorescence microscopy and by quantifying gene and protein expression. We found that DW had the greatest proliferative and least apoptotic and cytotoxic effects compared to other waveforms. DC, DW and CC stimulations resulted in a higher number of cells in S phase and G(2)/M phase as shown by cell cycle analysis. CC and DW caused more cells to invade collagen and showed increased MMP-2 and MT1-MMP expression. DC increased cellular migration in a scratch-wound assay and all ES waveforms enhanced expression of migratory genes with DC having the greatest effect. All ES treated cells showed similar progenitor potential as determined by MSC differentiation assay. All above findings were shown to be statistically significant (p<0.05). We conclude that ES can influence BMMSCs activities, especially DW and CC, which show greater invasion and higher cell proliferation compared to other types of ES. Application of DW or CC to the fracture site may help in the recruitment of BMMSCs to the wound that may enhance rate of bone healing at the fracture site.     

Efficient control of transient wave forms to prevent spreading depolarizations

In various neurological disorders spatio-temporal excitation patterns constitute examples of excitable behavior emerging from pathological pathways. During migraine, seizure, and stroke an initially localized pathological state can temporarily spread indicating a transition from non-excitable to excitable behavior. We investigate these transient wave forms in the generic FitzHugh-Nagumo (FHN) system of excitable media. Our goal is to define an efficient control minimizing the volume of invaded tissue. The general point of such a therapeutic optimization is how to apply control theory in the framework of structures in differential geometry by regarding parameter plane M of the FHN system as a differentiable manifold endowed with a metric. We suggest to equip M with a metric given by pharmacokinetic-pharmacodynamic models of drug receptor interaction.     

Rapid and transient decrease of N-myc expression in retinoic acid-induced differentiation of OTF9 teratocarcinoma stem cells.

The level of expression of N-myc in mouse teratocarcinoma stem cells is very high. Previous studies have shown that N-myc expression significantly decreases when the stem cells are subjected to long-term induction for differentiation by retinoic acid (RA). We found that in a stem cell line, OTF9, a steep yet transient decrease of N-myc expression takes place much earlier, immediately after induction by RA. To examine whether this decrease is responsible for differentiation, we constructed a gene, miwNmyc, to express N-myc cDNA constitutively and transformed OTF9 cells with this gene construct. Transformants under the constitutive expression of miwNmyc differentiated normally, as judged by morphological changes and by modulation of c-myc, Hox1.1, and laminin B1 expression. Therefore, transient decrease of N-myc expression may be the consequence of RA-induced differentiation, even though it occurs very early in the process. Alternatively, in addition to N-myc decrease, there may be redundant mechanisms which lead to OTF9 differentiation after induction by RA, so that suppression of N-myc decrease is bypassed by at least one other mechanism.     

Xanthine oxidase,an indicator of secretory differentiation in mammary cells

Elevated levels of xanthine oxidase were found in (1) lactating mouse mammary glands, compared with virgin and midpregnant glands; and (2) primary mouse mammary cells cultured on floating collagen gels, compared with non-secretory cells on attached gels. In primary culture, increase in xanthine oxidase activity above a basal level coincided with secretory activity as measured by casein production; intracellular levels of casein and xanthine oxidase showed a high degree of correspondence. It is suggested that xanthine oxidase levels can be used as an indicator of in vivo and in vitro secretory differentiation in mammary epithelial cells.     

BMP4 is required for the initial expression of MITF in melanocyte precursor differentiation from embryonic stem cells

Although the differentiation of melanoblasts to melanocytes is known to depend on many distinct factors, it is still poorly understood which factors lead to the induction of melanoblasts. To determine which factors might induce melanoblasts, we examined a set of candidate factors for their ability to induce expression of MITF, a master regulator of melanoblast development, in an ES cell-based melanocyte differentiation system. It appears that BMP4 is capable of inducing MITF expression in stem cells. In contrast, a number of other factors normally implicated in the development of the melanocyte lineage, including WNT1, WNT3a, SCF, EDN3, IGF1, PDGF, and RA, cannot induce MITF expression. Nevertheless, BMP4 alone does not allow MITF-expressing precursors to become differentiated melanocytes, but the addition of EDN3 further promotes differentiation of the precursors into mature melanocytes. Our results support a model in which BMP4 induces MITF expression in pluripotent stem cells and EDN3 subsequently promotes differentiation of these MITF expressing cells along the melanocyte lineage.     

Functional impairment of MSC induced by transient warming events: Correlation with loss of adhesion and altered cell size

Background

We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events.