Days Of Differentiation Research Articles

Using Rabbit Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Investigate Drug-Induced Fetal Heart Malformations

Introduction: Teratogenic events such as fetal heart malformations are serious and tragic drug-induced toxicities. Preclinical studies conducted during drug safety evaluation are intended to identify teratogenic risks using rodent and nonrodent models, largely based on the experience with thalidomide, which is teratogenic in humans and rabbits but refractory in rodents. Methods: To study the teratogenic properties of thalidomide in rabbit, a novel method was designed to develop cardiomyocytes (CMs) from rabbit induced pluripotent stem cells (rbiPSCs). Throughout the course of differentiation, rbiPSC-CMs were examined by real time polymerase chain reaction, high-content analysis, calcium flux measurements, and electrophysiology to confirm the cell's mature CM phenotype. The effects of thalidomide were then measured in each of the endpoints when present throughout differentiation. Results: As early as differentiation day (DD) 3 of the differentiation, rbiPSCs begin to express early CM markers (e.g., NXK2.5) and mesodermal markers (e.g., FOXF1). By DD 21, rbiPSC-CMs expressed messenger RNA (mRNA) and protein of mature CM genes, including TNNT2, HCN4, and KCNJ2. Spontaneous beating was observed by microscopy and electrophysiology as early as DD 8, and live cell calcium probes demonstrated pulsatile calcium flux. Thalidomide treatment to rbiPSC-CMs downregulated mRNA and protein of early and mature cardiac markers, suggesting suppressed differentiation. In addition, thalidomide treatment abrogated the onset of spontaneously beating cells. Conclusions: Taken together, these data indicate that rbiPSC-CMs can recapitulate thalidomide-induced cardiac teratogenicity observed in rabbits. Moving forward, this model can be deployed to investigate the underlying mechanisms of rabbit teratogenicity and better understand species-specific differences in drug-induced fetal heart malformations.

Culture Systems of Dissociated Mouse and Human Pluripotent Stem Cell-Derived Retinal Ganglion Cells Purified by Two-Step Immunopanning.

We aimed to establish purification and culture systems for retinal ganglion cells (RGCs) differentiated from mouse and human pluripotent stem cells (PSC) for in vitro and regenerative medicine studies. We used a two-step immunopanning method to purify RGCs from mouse and human PSC-derived three-dimensional (3D) retinal organoids. To assess the method, we purified RGCs from 3D retinal organoids derived from embryonic stem cells (ESCs) generated from Thy1-EGFP transgenic (TG) mice. In addition, 3D retinal organoids differentiated from human induced PSCs (iPSCs) were cultured for up to differentiation day (DD) 120, and RGCs were purified by immunopanning. RGC marker expressions were confirmed by immunostaining and reverse transcription-quantitative PCR. The purified RGCs were cultured, and neurite outgrowth was measured and analyzed using an IncuCyte Zoom system. Mouse RGCs purified from Thy1-EGFP TG mouse retinas and the ESC-derived 3D retinas could be maintained for approximately 2 to 3 weeks, expressing the markers BRN3B and SMI-312. Purified RGCs from human iPSC-derived retinal organoids expressed RGC markers and could be maintained for up to 4 weeks. The RGCs collected at DD 90 to 110 extended longer neurites than those collected at younger stages. We successfully purified RGCs from mouse and human PSC-derived 3D retinal organoids cultured for approximately 120 days. RGCs from older retinal organoids would be useful for neurite tracking. This method would be effective not only for studying the pathology of human RGC diseases but also for therapeutic drug studies and RGC transplantation.

Identification of Stage-Specific Gene Expression Signatures in Response to Retinoic Acid during the Neural Differentiation of Mouse Embryonic Stem Cells.

We have previously established a protocol for the neural differentiation of mouse embryonic stem cells (mESCs) as an efficient tool to evaluate the neurodevelopmental toxicity of environmental chemicals. Here, we described a multivariate bioinformatic approach to identify the stage-specific gene sets associated with neural differentiation of mESCs. We exposed mESCs (B6G-2 cells) to 10−8 or 10−7 M of retinoic acid (RA) for 4 days during embryoid body formation and then performed morphological analysis on day of differentiation (DoD) 8 and 36, or genomic microarray analysis on DoD 0, 2, 8, and 36. Three gene sets, namely a literature-based gene set (set 1), an analysis-based gene set (set 2) using self-organizing map and principal component analysis, and an enrichment gene set (set 3), were selected by the combined use of knowledge from literatures and gene information selected from the microarray data. A gene network analysis for each gene set was then performed using Bayesian statistics to identify stage-specific gene expression signatures in response to RA during mESC neural differentiation. Our results showed that RA significantly increased the size of neurosphere, neuronal cells, and glial cells on DoD 36. In addition, the gene network analysis showed that glial fibrillary acidic protein, a neural marker, remarkably up-regulates the other genes in gene set 1 and 3, and Gbx2, a neural development marker, significantly up-regulates the other genes in gene set 2 on DoD 36 in the presence of RA. These findings suggest that our protocol for identification of developmental stage-specific gene expression and interaction is a useful method for the screening of environmental chemical toxicity during neurodevelopmental periods.

Ethanol alters the expressions of c-Fos and myelin basic protein in differentiating oligodendrocytes

Myelination occurs in the central nervous system of the human fetus, adolescents, and young adults. Ethanol interferes with myelination in part by altering the composition of the myelin sheath. Here we show that ethanol also affected the expression of the transcription factor c-Fos in differentiating oligodendrocytes (OLGs). Central glial-4 OLG progenitors were induced to differentiate in the absence and presence of 100 mM ethanol, and ethanol-caused changes in the levels of c-Fos and myelin basic protein (MBP) were determined by Western blot analysis at selected developmental stages. The relatively high c-Fos level in progenitors did not immediately decrease to a low level at the onset of differentiation but displayed a downregulation at a later developmental stage. Ethanol delayed the developmental c-Fos downregulation maintaining c-Fos at a 45% higher level at 2 days of differentiation (DoD). Ethanol also decreased the rate of the burst of MBP expression that occurred between 1 and 2 DoD, reducing the MBP level by 47% at 2 DoD. The ethanol-caused delays of c-Fos downregulation and MBP upregulation were both blocked by the protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM). Likewise, treatment of OLGs with a low 5-nM concentration of the PKC activator by 12-O-tetradecanoylphorbol-13-acetate mimicked the ethanol effects on the expression of both proteins, effects that were also counteracted by BIM. The results indicate that ethanol-caused delays of the stage-specific c-Fos downregulation and the inhibition of MBP expression both occur through a PKC-mediated mechanism. The ethanol-caused delay in c-Fos downregulation may disrupt normal timing for expression of genes involved in OLG differentiation, and the inhibited MBP expression may alter the myelin sheath composition.