Regulation of cell death in mitotic neural progenitor cells by asymmetric distribution of prostate apoptosis response 4 (PAR-4) and simultaneous elevation of endogenous ceramide

Cell death and survival of neural progenitor (NP) cells are determined by signals that are largely unknown. We have analyzed pro-apoptotic signaling in individual NP cells that have been derived from mouse embryonic stem cells. NP formation was concomitant with elevated apoptosis and increased expression of ceramide and prostate apoptosis response 4 (PAR-4). Morpholino oligonucleotide-mediated antisense knockdown of PAR-4 or inhibition of ceramide biosynthesis reduced stem cell apoptosis, whereas PAR-4 overexpression and treatment with ceramide analogs elevated apoptosis. Apoptotic cells also stained for proliferating cell nuclear antigen (a nuclear mitosis marker protein), but not for nestin (a marker for NP cells). In mitotic cells, asymmetric distribution of PAR-4 and nestin resulted in one nestin(−)/PAR-4(+) daughter cell, in which ceramide elevation induced apoptosis. The other cell was nestin(+), but PAR-4(−), and was not apoptotic. Asymmetric distribution of PAR-4 and simultaneous elevation of endogenous ceramide provides a possible mechanism underlying asymmetric differentiation and apoptosis of neuronal stem cells in the developing brain

Comparing independent microarray studies: the case of human embryonic stem cells

BACKGROUND: Microarray studies of the same phenomenon in different labs often appear at variance because the published lists of regulated transcripts have disproportionately small intersections. We demonstrate that comparing studies by intersecting lists in this manner is methodologically flawed by reanalyzing three studies of the molecular signature of "stemness" in human embryonic stem cells. There are only 7 genes common to all three published lists, suggesting disagreement. RESULTS: Carefully reanalyzing all three together from the raw data we detect 111 genes upregulated and 95 downregulated in all three studies. The upregulated list was subject to rtRTPCR analysis and 75% of the genes were confirmed. CONCLUSION: Our findings show that the three studies have a substantial core of common genes, which is missed if only the published lists are examined. Combined analysis of multiple experiments can be a powerful way to distil coherent conclusions

A Time Course Analysis of the Electrophysiological Properties of Neurons Differentiated from Human Induced Pluripotent Stem Cells (iPSCs)

Many protocols have been designed to differentiate human embryonic stem cells (ESCs) and human induced pluripotent stem cells (iPSCs) into neurons. Despite the relevance of electrophysiological properties for proper neuronal function, little is known about the evolution over time of important neuronal electrophysiological parameters in iPSC-derived neurons. Yet, understanding the development of basic electrophysiological characteristics of iPSC-derived neurons is critical for evaluating their usefulness in basic and translational research. Therefore, we analyzed the basic electrophysiological parameters of forebrain neurons differentiated from human iPSCs, from day 31 to day 55 after the initiation of neuronal differentiation. We assayed the developmental progression of various properties, including resting membrane potential, action potential, sodium and potassium channel currents, somatic calcium transients and synaptic activity. During the maturation of iPSC-derived neurons, the resting membrane potential became more negative, the expression of voltage-gated sodium channels increased, the membrane became capable of generating action potentials following adequate depolarization and, at day 48–55, 50% of the cells were capable of firing action potentials in response to a prolonged depolarizing current step, of which 30% produced multiple action potentials. The percentage of cells exhibiting miniature excitatory post-synaptic currents increased over time with a significant increase in their frequency and amplitude. These changes were associated with an increase of Ca2+ transient frequency. Co-culturing iPSC-derived neurons with mouse glial cells enhanced the development of electrophysiological parameters as compared to pure iPSC-derived neuronal cultures. This study demonstrates the importance of properly evaluating the electrophysiological status of the newly generated neurons when using stem cell technology, as electrophysiological properties of iPSC-derived neurons mature over time

Directed Differentiation of Human Pluripotent Stem Cells to Microglia

Microglia, the immune cells of the brain, are crucial to proper development and maintenance of the CNS, and their involvement in numerous neurological disorders is increasingly being recognized. To improve our understanding of human microglial biology, we devised a chemically defined protocol to generate human microglia from pluripotent stem cells. Myeloid progenitors expressing CD14/CX3CR1 were generated within 30 days of differentiation from both embryonic and induced pluripotent stem cells (iPSCs). Further differentiation of the progenitors resulted in ramified microglia with highly motile processes, expressing typical microglial markers. Analyses of gene expression and cytokine release showed close similarities between iPSC-derived (iPSC-MG) and human primary microglia as well as clear distinctions from macrophages. iPSC-MG were able to phagocytose and responded to ADP by producing intracellular Ca 2+ transients, whereas macrophages lacked such response. The differentiation protocol was highly reproducible across several pluripotent stem cell lines

Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood

Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2–3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12015-015-9586-8) contains supplementary material, which is available to authorized users

Generation of iPSC lines from archived non-cryoprotected biobanked dura mater

Background: Induced pluripotent stem cells (iPSCs) derived from patients with neurodegenerative disease generally lack neuropathological confirmation, the gold standard for disease classification and grading of severity. The use of tissue with a definitive neuropathological diagnosis would be an ideal source for iPSCs. The challenge to this approach is that the majority of biobanked brain tissue was not meant for growing live cells, and thus was not frozen in the presence of cryoprotectants such as DMSO. Results: We report the generation of iPSCs from frozen non-cryoprotected dural tissue stored at −80°C for up to 11 years. This autopsy cohort included subjects with Alzheimer’s disease and four other neurodegenerative diseases. Conclusions: Disease-specific iPSCs can be generated from readily available, archival biobanked tissue. This allows for rapid expansion of generating iPSCs with confirmed pathology as well as allowing access to rare patient variants that have been banked

A dual SHOX2:GFP; MYH6:mCherry knockin hESC reporter line for derivation of human SAN-like cells

The sinoatrial node (SAN) is the primary pacemaker of the heart. The human SAN is poorly understood due to limited primary tissue access and limitations in robust in vitro derivation methods. We developed a dual SHOX2:GFP; MYH6:mCherry knockin human embryonic stem cell (hESC) reporter line, which allows the identification and purification of SAN-like cells. Using this line, we performed several rounds of chemical screens and developed an efficient strategy to generate and purify hESC-derived SAN-like cells (hESC-SAN). The derived hESC-SAN cells display molecular and electrophysiological characteristics of bona fide nodal cells, which allowed exploration of their transcriptional profile at single-cell level. In sum, our dual reporter system facilitated an effective strategy for deriving human SAN-like cells, which can potentially be used for future disease modeling and drug discovery.American Heart Association IncVoRSUNY DownstatePhysiology and PharmacologyN/