Expression of Plet1 controls interstitial migration of murine small intestinal dendritic cells

Under homeostatic conditions, dendritic cells (DCs) continuously patrol the intestinal lamina propria. Upon antigen encounter, DCs ini

Plant biotechnology

BACKGROUND: Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes. OBJECTIVE: We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha (hypoxia-inducible factor-1alpha) and the canonical Wnt pathway in this process. METHODS: Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture. RESULTS: At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway. CONCLUSION: Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis

Effects of hypoxia on HIF-1alpha and beta-catenin expression.

<p>A. Quantitative PCR showing significant increase of HIF-1α expression following 24 hours hypoxia alone compared to normoxic control. B. Representative Western blots of nuclear extracts from EBs, following 24 h or normoxia or hypoxia. Protein loading was examined by Western blotting against the nuclear protein RBAP46. C. Quantitative PCR showing significant increase of Mef2c expression following 24 hours hypoxia alone compared to normoxic control. All qPCR experiments (n = 4) were performed in 2 independent experiments.</p

Generation of iPS lines from skeletal muscle.

<p>(A). Muscle progenitors (i) and fibroblasts (ii) were isolated from skeletal muscle and used for reprogramming experiments. Four Representative iPS colonies obtained from muscle progenitor (iii, n = 2) and fibroblasts (iv, n = 2). (B). RT-PCR showing endogenous expression of pluripotency genes Nanog, Oct3/4 and Sox2 in reprogrammed iPS colonies. (C). Characterization of chromosomal content shows normal karyotyping (i) and differentiation in tissues from all 3 germ-layers endoderm, mesoderm and ectoderm (ii-adipocyte; iii-cartilage; iv-epidermis; v-ciliated epithelium; vi-neural rossettes) were identified in all 4 lines. (D). Quantification of expression of pluripotency genes after reprogramming for the 4 iPS lines generated, shown in different colors. Scale bar  =  50 um.</p

Time-course for expression of mesodermal marker brachyury and the cardiac progenitor marker Isl-1 (A) or for expression of differentiation/structural markers for cardiomyocytes (B) upon EB differentiation under normoxic conditions (n = 4, performed in 2 i

<p>Time-course for expression of mesodermal marker brachyury and the cardiac progenitor marker Isl-1 (A) or for expression of differentiation/structural markers for cardiomyocytes (B) upon EB differentiation under normoxic conditions (n = 4, performed in 2 independent experiments).</p

Diagramatic representation of the experimental approach for studies into the effects of hypoxia (A).

<p>Representative images of EBs under varying conditions, with 50(B). Observed beating rate (C) (n = 4, performed in 2 independent experiments).</p