Comprehensive Gene Expression Analysis of Human Embryonic Stem Cells during Differentiation into Neural Cells

Global gene expression analysis of human embryonic stem cells (hESCs) that differentiate into neural cells would help to further define the molecular mechanisms involved in neurogenesis in humans. We performed a comprehensive transcripteome analysis of hESC differentiation at three different stages: early neural differentiation, neural ectoderm, and differentiated neurons. We identified and validated time-dependent gene expression patterns and showed that the gene expression patterns reflect early ESC differentiation. Sets of genes are induced in primary ectodermal lineages and then in differentiated neurons, constituting consecutive waves of known and novel genes. Pathway analysis revealed dynamic expression patterns of members of several signaling pathways, including NOTCH, mTOR and Toll like receptors (TLR), during neural differentiation. An interaction network analysis revealed that the TGFβ family of genes, including LEFTY1, ID1 and ID2, are possible key players in the proliferation and maintenance of neural ectoderm. Collectively, these results enhance our understanding of the molecular dynamics underlying neural commitment and differentiation

Prospective Isolation of ISL1 + Cardiac Progenitors from Human ESCs for Myocardial Infarction Therapy

The LIM-homeodomain transcription factor ISL1 marks multipotent cardiac progenitors that give rise to cardiac muscle, endothelium, and smooth muscle cells. ISL1+ progenitors can be derived from human pluripotent stem cells, but the inability to efficiently isolate pure populations has limited their characterization. Using a genetic selection strategy, we were able to highly enrich ISL1+ cells derived from human embryonic stem cells. Comparative quantitative proteomic analysis of enriched ISL1+ cells identifiedALCAM(CD166) as a surface marker that enabled the isolation of ISL1+ progenitor cells. ALCAM+/ISL1+ progenitors are multipotent and differentiate into cardiomyocytes,endothelial cells, and smooth muscle cells. Transplantation of ALCAM+ progenitors enhances tissue recovery, restores cardiac function,and improves angiogenesis through activation of AKT-MAPK signaling in a rat model of myocardial infarction, based on cardiac MRI and histology. Our study establishes an efficient method for scalable purification of human ISL1+ cardiac precursor cells for therapeutic applications. (open access)

Androgen Regulates SARS-CoV-2 Receptor Levels and Is Associated with Severe COVID-19 Symptoms in Men.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has led to a global health crisis, and yet our understanding of the disease pathophysiology and potential treatment options remains limited. SARS-CoV-2 infection occurs through binding and internalization of the viral spike protein to angiotensin converting enzyme 2 (ACE2) on the host cell membrane. Lethal complications are caused by damage and failure of vital organs that express high levels of ACE2, including the lungs, the heart and the kidneys. Here, we established a high-throughput drug screening strategy to identify therapeutic candidates that reduce ACE2 levels in human embryonic stem cell (hESC) derived cardiac cells. Drug target analysis of validated hit compounds, including 5 alpha reductase inhibitors, revealed androgen signaling as a key modulator of ACE2 levels. Treatment with the 5 alpha reductase inhibitor dutasteride reduced ACE2 levels and internalization of recombinant spike receptor binding domain (Spike-RBD) in hESC-derived cardiac cells and human alveolar epithelial cells. Finally, clinical data on coronavirus disease 2019 (COVID-19) patients demonstrated that abnormal androgen states are significantly associated with severe disease complications and cardiac injury as measured by blood troponin T levels. These findings provide important insights on the mechanism of increased disease susceptibility in male COVID-19 patients and identify androgen receptor inhibition as a potential therapeutic strategy

Programming multicellular assembly with synthetic cell adhesion molecules.

Cell adhesion molecules are ubiquitous in multicellular organisms, specifying precise cell-cell interactions in processes as diverse as tissue development, immune cell trafficking and the wiring of the nervous system1-4. Here we show that a wide array of synthetic cell adhesion molecules can be generated by combining orthogonal extracellular interactions with intracellular domains from native adhesion molecules, such as cadherins and integrins. The resulting molecules yield customized cell-cell interactions with adhesion properties that are similar to native interactions. The identity of the intracellular domain of the synthetic cell adhesion molecules specifies interface morphology and mechanics, whereas diverse homotypic or heterotypic extracellular interaction domains independently specify the connectivity between cells. This toolkit of orthogonal adhesion molecules enables the rationally programmed assembly of multicellular architectures, as well as systematic remodelling of native tissues. The modularity of synthetic cell adhesion molecules provides fundamental insights into how distinct classes of cell-cell interfaces may have evolved. Overall, these tools offer powerful abilities for cell and tissue engineering and for systematically studying multicellular organization

ISL1 Protein Transduction Promotes Cardiomyocyte Differentiation from Human Embryonic Stem Cells

<div><h3>Background</h3><p>Human embryonic stem cells (hESCs) have the potential to provide an unlimited source of cardiomyocytes, which are invaluable resources for drug or toxicology screening, medical research, and cell therapy. Currently a number of obstacles exist such as the insufficient efficiency of differentiation protocols, which should be overcome before hESC-derived cardiomyocytes can be used for clinical applications. Although the differentiation efficiency can be improved by the genetic manipulation of hESCs to over-express cardiac-specific transcription factors, these differentiated cells are not safe enough to be applied in cell therapy. Protein transduction has been demonstrated as an alternative approach for increasing the efficiency of hESCs differentiation toward cardiomyocytes.</p> <h3>Methods</h3><p>We present an efficient protocol for the differentiation of hESCs in suspension by direct introduction of a LIM homeodomain transcription factor, Islet1 (ISL1) recombinant protein into the cells.</p> <h3>Results</h3><p>We found that the highest beating clusters were derived by continuous treatment of hESCs with 40 µg/ml recombinant ISL1 protein during days 1–8 after the initiation of differentiation. The treatment resulted in up to a 3-fold increase in the number of beating areas. In addition, the number of cells that expressed cardiac specific markers (cTnT, CONNEXIN 43, ACTININ, and GATA4) doubled. This protocol was also reproducible for another hESC line.</p> <h3>Conclusions</h3><p>This study has presented a new, efficient, and reproducible procedure for cardiomyocytes differentiation. Our results will pave the way for scaled up and controlled differentiation of hESCs to be used for biomedical applications in a bioreactor culture system.</p> </div

Daily qRT-PCR analysis in aggregate differentiation of hESCs.

<p>Undifferentiated aggregates of hESCs were treated by Activin A for 1 day and then for 4 days by BMP4. At day 5, the aggregates were plated without cytokines. The data show the maximum expression of the mesoendodermal marker, <i>Brachyury</i>, one day after Activin A treatment (day 2 after differentiation initiation). By continuing differentiation with BMP4 for the next 4 days <i>Isl1</i>, a marker of precardiac mesoderm, and <i>Actinin</i> were reached to their highest expression level. <i>Isl1</i> expression was remained at high level for the next 3 days and by decreasing its expression, <i>Mef2c</i>, a cardiac progenitor marker showed its maximum expression and after that, other cardiac progenitor genes, <i>Gata4 Nkx2.5</i> and <i>Tbx5</i> reached to their highest expression level respectively. Finally, the expression of <i>MHC</i> and <i>cTnT</i>, which are structural cardiomyocytes markers, got to maximum level (Fig. 1). These data shows that 3-dimentional structures of the cells are very important for cardiac differentiation and aggregated differentiation method enhances cardiac differentiation and functionality.Target genes were normalized by the reference gene <i>Gapdh</i>. The relative expression was calculated by dividing the normalized target gene expression of the treated sample with that of the undifferentiated state (day 0). All data represented as log2-linear plots. All data are statistically significant otherwise marked with “ns” (P>0.05).</p

Optimization of the effect of rISL1 protein on hESCs.

<p>(A) To evaluate the effect of discontinuous (2 h/day) or continuous rISL1 protein addition on hESCs (Royan H5) differentiation, cells were treated continuously or discontinuously from days 1–8 post initiation of differentiation. <i>Isl1</i> qRT-PCR analysis of differentiated cells at day 8 showed higher significant endogenous <i>Isl1</i> expression in hESCs in the continuous protocol.Thus continuous treatment was applied in the next steps. )* : P<0.05((B) To determine the best concentration of rISL1 protein for cardiac differentiation, cells were treated with four different concentrations of recombinant protein: 10, 20, 30, and 40 µg/ml in continuous treatment of hESCs during days 1–8 after initiation of differentiation. During differentiation, cells that were treated by 10 and 20 µg/ml rISL1 protein were morphologically similar to hematopoietic and endothelial progenitors, while the 30 and 40 µg/ml concentrations showed cardiomyocyte and muscular appearances. It seems that 30 and 40 µg/ml rISL1 protein are better concentrations for cardiac differentiation. )* : P<0.05((C) qRT-PCR analysis of differentiated cells at day 8 by different concentrations of rISL1 also showed that 40 µg/ml of the rISL1 protein induced more endogenous <i>Isl1</i>, but less <i>Mef2c</i> and <i>Nkx2.5</i> expressions. )* : P<0.05((D) Schematic diagram of the differentiation protocol by the addition of rISL1 protein (40 µg/ml), which was added after induction with Activin A (days 1–8). qRT-PCR analysis of endogenous <i>Isl1</i> expression in hESCs demonstrated that treated cells expressed higher significant endogenous <i>Isl1</i> than the untreated control. )* : P<0.05((E) The percentage of beating clusters in continuous treatment of hESCs by 40 µg/ml rISL1 protein during days 1–8 after differentiation initiation in comparison with the control (vehicle-treated) group. The percentage of beating clusters in the rISL1-treated group was significantly higher than the untreated group at day 14 after plating (75±10% vs. 20±2.5%). )* : P<0.05((F) rISL1 treatment resulted in a 3.2±0.5 fold increase in the number of beating areas in comparison with untreated control group. rISL1 also caused a 2.2±0.4 fold increase in the other hESC line, Royan H6, which shows the reproducibility of this protocol for another hESC line. )* : P<0.05((G) In order to assess the expression of cardiac-specific genes, we collected samples at 3 stages: day 3 after plating (the day of rISL1 removal); day 14 after plating (day of maximum beating); and day 20 after plating (day that beating decreased and cells were mature) by qRT-PCR in two hESC lines. Target genes were normalized by the reference gene <i>Gapdh</i>. The relative expression was calculated by dividing the normalized target gene expression of treated hESCs with rISL1 protein and elution buffer (as control) with that of the undifferentiated state (day 0). All data are statistically significant in comparison with undifferentiated state (day 0) otherwise marked with “ns” (ns: P>0.05). a: P<0.05 in comparison with control group (elution buffer treated group). All data were represented as log2-linear plots.</p