Induction Of Embryonic Stem Cells Research Articles

BCR::ABL1 -Dependent Promotion of Hematopoietic Stem Cells (HSC) and Progenitors in an Inducible Embryonic Stem Cell (ESC) Model

Our current understanding of the effect of BCR::ABL1 oncogene in leukemia development largely relies on studies using in vitro and in vivo leukemogenesis models. Among them, in vivo models using retrovirus-mediated BCR-ABL gene transfer in murine embryonic stem cell (ESC)-derived hematopoietic cells suggested that the enforced expression of BCR::ABL1 was sufficient to confer them an in vivo long-term repopulation potential. However, in these models, the unique BCR::ABL1 dependence of the generation of this potential has not been shown. Our study aimed at getting a better understanding of the early events regulated by BCR::ABL1 during the course of ESC differentiation without retroviral-mediated gene transfer. The elucidation of these early events could also be of major interest in therapy as generation of bona fide HSCs from normal ESC or iPSC remains a major challenge. To this end, we engineered a unique Tet-ON inducible system in which BCR::ABL1(p210) expression level can be tightly controlled using doxycycline (Dox). As compared to previously reported ESC-derived models, our system contains a single copy of BCR::ABL1 inserted into the murine Col1a1 locus under the control of Tetracycline operator, is sensitive to Dox, leakproof and reversible. The first striking results was the timing of Dox-inducible BCR-ABL expression during the course of ESC differentiation, impacting both the frequency and emergence of hematopoietic progenitors. Indeed, the expression of BCR::ABL1 at day+5 favors significantly the emergence and maintenance of immature hematopoietic progenitors. Importantly, these cells can self-renew only in the presence of Dox. Several hematopoietic stem-like cell lines were established and two of them (HSCL-A and HSCL-C) have been explored in detail. A major BCR-ABL-dependent proliferative ability was documented in both (Figure 1D). Both expressed Lin-Sca-1+c-Kit+CD150+CD48- and eSLAM, corresponding to phenotypically identified adult mouse HSCs. Importantly, these cells exhibited a normal myeloid and erythroid differentiation capacity in CFC assays, giving rise also to CFU-GEMM. We then evaluated the ability of HSCL to self-renew using long-term culture-initiating cell (LTC-IC) assays. To this purpose, EPCR+CD45+/loCD48-CD150+ (ESLAM) cells were sorted at a single cell level and co-cultured on a supportive AFT024 feeder layers for 4 weeks. LTC-IC-derived progenitors were then assessed using CFC assays. The frequency of ESLAM LTC-ICs was 0.53 from adult bone marrow and 0.52 from HSCL lines demonstrating their self-renewal ability. To better characterize HSCL cell lines, we sorted LSK cell population from HSCL#A, HSCL#C, E14.5 fetal liver and murine adult bone marrow and analyzed their transcriptome signatures by RNA-Seq. Unsupervised principal component analysis with the 1333 genes exhibiting the highest variability revealed that HSCL cell lines clustered together and exhibited distinct molecular signature from fetal and adult LSK. This stratification was validated by clustering with the 50 best variable genes. Among them, we found 17 genes highly expressed in HSCL cell lines associated with HSC quiescence (such as NupR1). GSEA analyses confirmed an enrichment of several cell cycle genes as well as genes involved in unfolded protein response (UPR) and autophagy (p <0.001). Thus, we report here for the first time, a unique BCR-ABL inducible ESC-derived hematopoiesis model which demonstrates the ability of BCR-ABL expression to induce a proliferative signal for extended periods of time in highly primitive hematopoietic cells. This novel tool can be used to dissect CML ontogeny both in vitro and in vivo and perform drug screening at the most primitive stem cell level. It can also be of major interest to uncover molecular events and signaling pathways activated during normal ESC-derived hematopoiesis. Such knowledge will open up new avenues to differentiate human iPSC towards bona fide HSCs for potential clinical applications. Figure 1. Establishment of ES cell-derived hematopoietic cell lines expressing BCR::ABLp210.(A) Representation of the timeline of ES cell differentiation protocol and doxycycline induction. (B) HSCL#A cell morphology. (C) MGG staining of HSCL#A cells. (D) Proliferation curves of HSCL#A and HSCL#C cell lines over 40 days in culture. (E)Kinetics of the effect of doxycycline removal on HSCL#A cells cultured on OP9 cells for 4 consecutive days. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

SUMO conjugation susceptibility of Akt/protein kinase B affects the expression of the pluripotency transcription factor Nanog in embryonic stem cells.

Akt/PKB is a kinase involved in the regulation of a wide variety of cell processes. Its activity is modulated by diverse post-translational modifications (PTMs). Particularly, conjugation of the small ubiquitin-related modifier (SUMO) to this kinase impacts on multiple cellular functions, such as proliferation and splicing. In embryonic stem (ES) cells, this kinase is key for pluripotency maintenance. Among other functions, Akt is known to promote the expression of Nanog, a central pluripotency transcription factor (TF). However, the relevance of this specific PTM of Akt has not been previously analyzed in this context. In this work, we study the effect of Akt1 variants with differential SUMOylation susceptibility on the expression of Nanog. Our results demonstrate that both, the Akt1 capability of being modified by SUMO conjugation and a functional SUMO conjugase activity are required to induce Nanog gene expression. Likewise, we found that the common oncogenic E17K Akt1 mutant affected Nanog expression in ES cells also in a SUMOylatability dependent manner. Interestingly, this outcome takes places in ES cells but not in a non-pluripotent heterologous system, suggesting the presence of a crucial factor for this induction in ES cells. Remarkably, the two major candidate factors to mediate this induction, GSK3-β and Tbx3, are non-essential players of this effect, suggesting a complex mechanism probably involving non-canonical pathways. Furthermore, we found that Akt1 subcellular distribution does not depend on its SUMOylatability, indicating that Akt localization has no influence on the effect on Nanog, and that besides the membrane localization of E17K Akt mutant, SUMOylation is also required for its hyperactivity. Our results highlight the impact of SUMO conjugation in the function of a kinase relevant for a plethora of cellular processes, including the control of a key pluripotency TF.

Abstract 323: A Novel Human Specific Long Noncoding RNA PG1 Controls Human Cardiogenesis via Regulating Beta-catenin Activity

Long noncoding RNAs (lncRNAs) can regulate gene expression, signaling pathway, cellular and tissue functions at various stages. Recently, accumulating evidences suggest that lncRNAs play important roles in cell fate specification and organ development. We found hundreds of lncRNAs, which show lineage-specific expressions in human embryonic stem (ES) cell-derived multipotential cardiovascular progenitor cells (MCPs), cardiomyocytes (CMs), smooth muscle cells (SMs) or endothelial cells (ECs), by using whole transcriptome sequencing. Among MCP-specific lncRNAs, we identified a novel human lncRNA, PG1, which controls cardiovascular differentiation from human pluripotent stem cells. We found PG1 could regulate the early segregation of nascent mesoderm cells towards cardiac verse hematopoietic fate. Knockout of PG1 in human induced pluripotent stem (iPS) cells and H1 ES cells by CRISPR/Cas9 significantly increased hematopoietic differentiation from pluripotent stem cells when compared with wild type controls. Furthermore, our data suggest PG1 could bind beta-catenin protein to modulate Wnt signaling activity to regulate nascent mesoderm specification. In summary, we identified a novel human specific lncRNA, PG1, which plays an essential role in early stage human heart development.

Differentiation of embryonic stem cells into inner ear vestibular hair cells using vestibular cell derived-conditioned medium

Vestibular hair cells (V–HCs) in the inner ear have important roles and various functions. When V–HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V–HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V–HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V–HCs.

Mouse embryonic stem cells: The establishment of the system to produce differentiated cell types in vitro

During the last few years, embryonic stem (ES) cells have been a new tool in cell biology which is very promising for the scientific community to develop new cell therapies. ES cells are the only cell type that can differentiate into derivates of the three primary germ layers, not only in vivo but also, and most important, in vitro . This so-called pluripotency has resulted in the field of stem cell technology going into overdrive, and the establishment of many protocols for optimal maintenance, culture, genetic transfection and in vitro differentiation. The first pluripotent cells had been derived from teratocarcinomas, maligne tumors, and showed some disadvantages. Therefore later embryonic stem cells, and now adult stem cells are getting special attention from the scientists. In this study, we established for the first time in our country, the prolonged culture of undifferentiated ES cells in vitro and the pointed induction of cell differentiation into specific cell types. It is the result of an international collaboration program supported by Brazil and Germany, CAPES and DAAD (PROBRAL). The well-established routine should be clearly demonstrated by the continuous culture and propagation of several mouse ES lines in vitro under specific culture conditions preventing differentiation. On the other hand, these ES cells were exposed to defined differentiation induction systems to obtain specialized cells as cardiogenic, neurogenic and myogenic cell types. This demonstrates the successful procedure to induce ES cell line differentiation. In this study, we established both routine systems, with and without differentiation. This results gave us competence and possibility to develop a series of different scientific approaches.

Chondrogenic differentiation of embryonic stem cells using mechanotransductive 3-D PDMS scaffolds

Embryonic stem cells (ESCs) are an ideal source for cell therapy and regenerative medicine. ESCs must be differentiated prior to their use for therapeutic applications. However, homogeneous differentiation of ESCs in vitro has proven to be challenging. We hypothesized that both biological and mechanical cues contribute to the specific differentiation of ESCs in vivo . This was tested by mimicking the in vivo microenvironment to differentiate ESCs into chondrogenic lineage using highly elastic polydimethylsiloxane (PDMS) scaffolds and the application of mechanical compression in vitro . ESCs seeded in PDMS scaffolds subjected to static compressive stress resulted in significant upregulation of genes, Sox9, Col2, and Acan , involved in early chondrogenic differentiation . However, the compressive stress did not affect expression of the late hypertrophic markers, Runx2 , Col10, and Mmp13 , signifying induction of ESCs into early chondroprogenitors. Application of mechanical stress increased expression of mechanical signaling genes, Rhoa , Yap, and Taz . The chondroinductive role of Rhoa was confirmed by the inhibition of RhoA signaling by CCG-1423, which resulted in decreased transcriptional and translational expression of chondrogenic markers. Based on these findings, we proposed a strategy for compression induced chondrogenic differentiation in mechanotransductive 3-D PDMS scaffolds.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

Abstract 1916: Peptidylarginine deiminase IV (PADI4) is a novel tumor suppressor that may contribute to regulation of breast cancer stem cell dynamics

Abstract Breast cancer is a global problem that accounts for almost a fourth of all cancers in women. Despite the many therapeutic strategies currently in use, treated patients often suffer from cancer relapse and metastasis due to the presence of a subpopulation of tumorigenic cells capable of self-renewal and termed cancer stem cells (CSCs). To address the properties of this critical sub-population, we utilized our recently-developed lentiviral CSC reporter carrying an Oct4/Sox2 response element coupled to a green fluorescent protein (SORE6-GFP) to detect and isolate the CSC population in a low-grade ER+ human breast cancer model, MCF10Ca1h, by FACS. Whole transcriptome sequencing analysis (RNA-seq) on the sorted cells identified Peptidylarginine deiminase IV (PADI4) as differentially expressed between CSCs and non-CSCs. PADI4 was recently shown to regulate citrullination of histone H1, displacing it from chromatin and resulting in global chromatin decondensation and pluripotency induction in embryonic stem cells (Christophorou, et al., 2014, Nature 507:104). Thus, we hypothesized that PADI4 may also regulate the stemness phenotype in breast cancer CSCs, likely through effects on chromatin structure. We showed that PADI4 protein expression is higher in luminal breast cancer cell lines than other subtypes, and we did shRNA knockdown (KD) of PADI4 in the luminal MCF10Ca1h model. PADI4 KD increased tumorosphere formation and clonogenicity indicating increased CSC population/activity. Additionally, MCF10Ca1h cells treated with a PADI4 selective inhibitor showed an increase in CSCs as assessed using the SORE6-GFP stem cell reporter. In vivo limiting dilution studies demonstrate that PADI4 KD increased tumor incidence at low cell inoculum, reflecting a statistically significant 2-4 fold increase in CSCs upon PADI4 KD. Our findings suggest that PADI4 may function as a tumor suppressor in luminal breast cancer. In support of this conclusion, deep deletions of PADI4 locus are found in 1% of human breast cancers in the TCGA datasets. Analysis of the effect of PADI4 KD on chromatin organization in the CSC and non-CSC compartments is ongoing. Citation Format: Nellie Moshkovich. Peptidylarginine deiminase IV (PADI4) is a novel tumor suppressor that may contribute to regulation of breast cancer stem cell dynamics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1916. doi:10.1158/1538-7445.AM2017-1916

Advance in methods of induced-embryonic stem cell differentiation to retinal cells

Retinal degenerative disease is the leading cause of visual loss, the mechanism is not clear and has no effective treatment method.In recent years, the field of stem cell research has made great progress.Stem cells have the potential of differentiating into all the body cells.Embryonic stem cells (ESCs) can be used to differentiate a variety of retinal cells, which has brought a new promise for the treatment of retinal degeneration disease.However, the most important step of this treatment is how to differentiate ESCs into photoreceptor cells and retinal pigment epithelium (RPF) cells.In this paper, we introduced the recent progress in the differentiation methods of ESCs into retinal cells, which contains spontaneous differentiation method, co-culture method, growth factors and small molecules induced method, adherent monoculture method, three dimensional differentiation method. Key words: Retinal degenerative disease; Embryonic stem cells; Differentiation; Retinal cells

Forced expression of Hnf1b/Foxa3 promotes hepatic fate of embryonic stem cells

Embryonic stem (ES) cell-derived hepatocytes have the potential to be used for basic research, regenerative medicine, and drug discovery. Recent reports demonstrated that in addition to conventional differentiation inducers such as chemical compounds and cytokines, overexpression of lineage-specific transcription factors could induce ES cells to differentiate to a hepatic fate. Here, we hypothesized that lentivirus-mediated inducible expression of hepatic lineage transcription factors could enhance mouse ES cells to hepatocyte-like cells. We screened the effects of candidate transcription factors Hnf1b, Hnf1a, Hnf4a, Foxa1, Foxa3 and Hex, and determined that the combination of Hnf1b/Foxa3 promoted expression of several hepatic lineage-specific markers and proteins, in addition to glycogen storage, ICG uptake, and secretion of albumin and urea. The differentiated cells were engraftable and expressed albumin when transplanted into a carbon tetrachloride-injured mouse model. These results demonstrated the crucial role of Hnf1b and Foxa3 in hepatogenesis in vitro and provided a valuable tool for the efficient differentiation of HLCs from ES cells.

Zscan10 is dispensable for maintenance of pluripotency in mouse embryonic stem cells

Zinc finger and SCAN domain-containing 10 (Zscan10, also known as Zfp206) encodes a transcription factor that has been reported to be involved in the maintenance of pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Zscan10 using the Cre-loxP system and analyzed its function. We succeeded in establishing Zscan10-null ES cells and confirmed their pluripotency by the generation of chimeric embryos. Our results clearly indicate that Zscan10 is dispensable for the ability of self-renewal and differentiation in ES cells.

Role of TRPV1 in the Differentiation of Mouse Embryonic Stem Cells into Cardiomyocytes.

Cytosolic Ca2+ ([Ca2+]i) is an important signal that regulates cardiomyocyte differentiation during cardiogenesis. TRPV1 is a Ca2+-permeable channel that is expressed in cardiomyocytes. In the present study, we utilized mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) as a model to investigate the functional role of TRPV1 in cardiomyocyte differentiation. Induction of embryonic stem cells into cardiomyocytes was achieved using embryoid body (EB)-based differentiation method. Quantitative PCRs showed an increased TRPV1 expression during the differentiation process. In [Ca2+]i measurement study, application of TRPV1 agonists, capsaicin and camphor, elicited a [Ca2+]i rise in mESC-CMs, the effect of which was abolished by TRPV1-shRNA. In functional study, treatment of EBs with TRPV1 antagonists (capsazepine and SB366791) and TRPV1-shRNA reduced the size of the EBs and decreased the percentage of spontaneously beating EBs. TRPV1 antagonists and TRPV1-shRNA also suppressed the expression of cardiomyocyte marker genes, including cardiac actin, c-TnT, c-TnI, and α-MHC. Taken together, this study demonstrated an important functional role of TRPV1 channels in the differentiation of mESCs into cardiomyocytes.

Generation of HSC-Like Cells from Human Embryonic Stem Cells by Inhibition of TGF-βR2 Signaling

The efficient induction of embryonic stem cells into hematopoietic stem cells (HSCs) requires understanding the pro or inhibitory regulatory networks, which are involved in these pathways. Transforming growth factor β family is a super family of growth factors such as TGF-β1, TGF-β2, TGF-β3, and some other cytokines. Previous works indicate that some of TGF-β subfamilies have negative regulatory effect during hematopoietic differentiation and HSC proliferation. The inhibitory effects of TGF-β1 in early stage of differentiation of endothelial and hematopoietic cells have been shown. Therefore, in the present study, in order to evaluate the promotion of hematopoietic differentiation, the activities of TGF-β regulatory factor were reduced through knockdown of TGF-βR2 expression. The ESCs were transduced by Lentivirus carrying SiTGF-βR2 and its scramble separately. Green fluorescent protein (GFP) was used as a marker to monitor gene expression. Six days post transduction GFP+ colonies were used for EB generation and the next day, the same size EBs were transferred in differentiation media involved in defining cytokines. The expression level of hematopoietic-specific genes was evaluated in 0, 7, and 15-days of differentiation. The real-time PCR analysis in 15 day EBs showed 2–3-folds increase in SiTGF-βR2 as compared with the scramble. Therefore, this finding may be confirmed if TGF receptor silencing can reduce the inhibitory effect of TGF-β during early hematopoietic differentiation.

Nr0b1 is a negative regulator of Zscan4c in mouse embryonic stem cells.

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1, also known as Dax1) is regarded as an important component of the transcription factor network that governs pluripotency in mouse embryonic stem (ES) cells. Here we generated inducible knockout ES cells for Nr0b1 using the Cre-loxP system and analyzed its precise function. We succeeded in establishing the Nr0b1-null ES cells and confirmed their pluripotency by showing their contribution to chimeric embryos. However, they proliferated slowly with over-expression of 2-cell stage specific transcripts including Zscan4c, which is known to be involved in telomere elongation in ES cells. We revealed that over-expression of Zscan4c prevents normal self-renewal by inducing arrest at G2 phase followed by cell death and that Nr0b1 directly represses the Zscan4c promoter. These data indicated that Nr0b1 is not essential to maintain pluripotency but is involved in the proper activation of 2-cell specific transcripts for self-renewal.

Pax6- and Six3-mediated induction of lens cell fate in mouse and human ES cells.

Embryonic stem (ES) cells provide a potentially useful in vitro model for the study of in vivo tissue differentiation. We used mouse and human ES cells to investigate whether the lens regulatory genes Pax6 and Six3 could induce lens cell fate in vitro. To help assess the onset of lens differentiation, we derived a new mES cell line (Pax6-GFP mES) that expresses a GFP reporter under the control of the Pax6 P0 promoter and lens ectoderm enhancer. Pax6 or Six3 expression vectors were introduced into mES or hES cells by transfection or lentiviral infection and the differentiating ES cells analyzed for lens marker expression. Transfection of mES cells with Pax6 or Six3 but not with other genes induced the expression of lens cell markers and up-regulated GFP reporter expression in Pax6-GFP mES cells by 3 days post-transfection. By 7 days post-transfection, mES cell cultures exhibited a>10-fold increase over controls in the number of colonies expressing γA-crystallin, a lens fiber cell differentiation marker. RT-PCR and immunostaining revealed induction of additional lens epithelial or fiber cell differentiation markers including Foxe3, Prox1, α- and β-crystallins, and Tdrd7. Moreover, γA-crystallin- or Prox1-expressing lentoid bodies formed by 30 days in culture. In hES cells, Pax6 or Six3 lentiviral vectors also induced lens marker expression. mES cells that express lens markers reside close to but are distinct from the Pax6 or Six3 transduced cells, suggesting that the latter induce nearby undifferentiated ES cells to adopt a lens fate by non-cell autonomous mechanisms. In sum, we describe a novel mES cell GFP reporter line that is useful for monitoring induction of lens fate, and demonstrate that Pax6 or Six3 is sufficient to induce ES cells to adopt a lens fate, potentially via non-cell autonomous mechanisms. These findings should facilitate investigations of lens development.

Fetal stem cell transplantation: Past, present, and future

Since 1928, human fetal tissues and stem cells have been used worldwide to treat various conditions. Although the transplantation of the fetal midbrain substantia nigra and dopaminergic neurons in patients suffering from Parkinson's disease is particularly noteworthy, the history of other types of grafts, such as those of the fetal liver, thymus, and pancreas, should be addressed as there are many lessons to be learnt for future stem cell transplantation. This report describes previous practices and complications that led to current clinical trials of isolated fetal stem cells and embryonic stem (ES) cells. Moreover, strategies for transplantation are considered, with a particular focus on donor cells, cell processing, and the therapeutic cell niche, in addition to ethical issues associated with fetal origin. With the advent of autologous induced pluripotent stem cells and ES cells, clinical dependence on fetal transplantation is expected to gradually decline due to lasting ethical controversies, despite landmark achievements.

Regulation of transcription by the MLL2 complex and MLL complex–associated AKAP95

Although histone H3 lysine 4 (H3K4) methylation is widely associated with gene activation, direct evidence for its causal role in transcription, through specific MLL family members, is scarce. Here we have purified a human MLL2 (Kmt2b) complex that is highly active in H3K4 methylation and chromatin transcription in a cell-free system. This effect requires SAM and intact H3K4, establishing a direct and causal role for MLL2-mediated H3K4 methylation in transcription. We then show that human AKAP95, a chromatin-associated protein, is physically and functionally associated with the DPY30–MLL complexes and directly enhances their methyltransferase activity. Ectopic AKAP95 stimulates expression of a chromosomal reporter in synergy with MLL1 or MLL2, whereas AKAP95 depletion impairs retinoic acid-mediated gene induction in embryonic stem cells. These results demonstrate an important role for AKAP95 in regulating histone methylation and gene expression, particularly during cell fate transitions.

Utilization of transgenic models in the evaluation of osteogenic differentiation of embryonic stem cells

Previous studies reported that embryonic stem cells (ESCs) can be induced to differentiate into cells showing a mature osteoblastic phenotype by culturing them under osteo-inductive conditions. It is probable that osteogenic differentiation requires that ESCs undergo differentiation through an intermediary step involving a mesenchymal lineage precursor. Based on our previous studies indicating that adult mesenchymal progenitor cells express α-smooth muscle actin (αSMA), we have generated ESCs from transgenic mice in which an αSMA promoter directs the expression of red fluorescent protein (RFP) to mesenchymal progenitor cells. To track the transition of ESC-derived MSCs into mature osteoblasts, we have utilized a bone-specific fragment of rat type I collagen promoter driving green fluorescent protein (Col2.3GFP). Following osteogenic induction in ESCs, we have observed expression of alkaline phosphatase (ALP) and subsequent mineralization as detected by von Kossa staining. After 1 week of osteogenic induction, ESCs begin to express αSMARFP. This expression was localized to the peripheral area encircling a typical ESC colony. Nevertheless, these αSMARFP positive cells did not show activation of the Col2.3GFP promoter, even after 7 weeks of osteogenic differentiation in vitro. In contrast, Col2.3GFP expression was detected in vivo, in mineralized areas following teratoma formation. Our results indicate that detection of ALP activity and mineralization of ESCs cultured under osteogenic conditions is not sufficient to demonstrate osteogenic maturation. Our study indicates the utility of the promoter-visual transgene approach to assess the commitment and differentiation of ESCs into the osteoblast lineage.

TGF-β/Smad2/3 Signaling Directly Regulates Several miRNAs in Mouse ES Cells and Early Embryos

The Transforming Growth Factor-β (TGF-β) signaling pathway is one of the major pathways essential for normal embryonic development and tissue homeostasis, with anti-tumor but also pro-metastatic properties in cancer. This pathway directly regulates several target genes that mediate its downstream functions, however very few microRNAs (miRNAs) have been identified as targets. miRNAs are modulators of gene expression with essential roles in development and a clear association with diseases including cancer. Little is known about the transcriptional regulation of the primary transcripts (pri-miRNA, pri-miR) from which several mature miRNAs are often derived. Here we present the identification of miRNAs regulated by TGF-β signaling in mouse embryonic stem (ES) cells and early embryos. We used an inducible ES cell system to maintain high levels of the TGF-β activated/phosphorylated Smad2/3 effectors, which are the transcription factors of the pathway, and a specific inhibitor that blocks their activation. By performing short RNA deep-sequencing after 12 hours Smad2/3 activation and after 16 hours inhibition, we generated a database of responsive miRNAs. Promoter/enhancer analysis of a subset of these miRNAs revealed that the transcription of pri-miR-181c/d and the pri-miR-341∼3072 cluster were found to depend on activated Smad2/3. Several of these miRNAs are expressed in early mouse embryos, when the pathway is known to play an essential role. Treatment of embryos with TGF-β inhibitor caused a reduction of their levels confirming that they are targets of this pathway in vivo. Furthermore, we showed that pri-miR-341∼3072 transcription also depends on FoxH1, a known Smad2/3 transcription partner during early development. Together, our data show that miRNAs are regulated directly by the TGF-β/Smad2/3 pathway in ES cells and early embryos. As somatic abnormalities in functions known to be regulated by the TGF-β/Smad2/3 pathway underlie tumor suppression and metastasis, this research also provides a resource for miRNAs involved in cancer.

Baicalin Maintains Late-Stage Functional Cardiomyocytes in Embryoid Bodies Derived from Murine Embryonic Stem Cells

Background/Aims: Low efficiency of cardiomyocyte (CM) differentiation from embryonic stem (ES) cells limits their therapeutic use. The objective of this study was to investigate the effect of baicalin, a natural flavonoid compound, on the in vitro cardiac differentiation of murine ES cells. Methods: The induction of ES cells into cardiac-like cells was performed by embryoid body (EB)-based differentiation method. The electrophysiological properties of the ES cell-derived CMs (ES-CMs) were measured by patch-clamp. The biomarkers of ES-CMs were determined by quantitative RT-PCR and immunofluorescence. Results: Continuous baicalin treatment decreased the size of EBs, and increased the proportion of α-actinin-positive CMs and transcript level of cardiac specific markers in beating EBs by inducing cell death of non-CMs. Baicalin increased the percentage of working ES-CMs which had typical responses to β-adrenergic and muscarinic stimulations. Conclusion: Baicalin maintains the late-stage functional CMs in EBs derived from murine ES cells. This study describes a new insight into the various biological effects of baicalin on cardiac differentiation of pluripotent stem cells.