Evaluation of commonly used ectoderm markers in iPSC trilineage differentiation.

Patient-derived induced pluripotent stem cells (iPSCs) have become a promising resource for exploring genetics of complex diseases, discovering new drugs, and advancing regenerative medicine. Increasingly, laboratories are creating their own banks of iPSCs derived from diverse donors. However, there are not yet standardized guidelines for qualifying these cell lines, i.e., distinguishing between bona fide human iPSCs, somatic cells, and imperfectly reprogrammed cells. Here, we report the establishment of a panel of 30 iPSCs from CD34+ peripheral blood mononuclear cells, of which 10 were further differentiated in vitro into all three germ layers. We characterized these different cell types with commonly used pluripotent and lineage specific markers, and showed that NES, TUBB3, and OTX2 cannot be reliably used as ectoderm differentiation markers. Our work highlights the importance of marker selection in iPSC authentication, and the need for the field to establish definitive standard assays

Isogenic Cellular Systems Model the Impact of Genetic Risk Variants in the Pathogenesis of Type 1 Diabetes

At least 57 independent loci within the human genome confer varying degrees of risk for the development of type 1 diabetes (T1D). The majority of these variants are thought to contribute to overall genetic risk by modulating host innate and adaptive immune responses, ultimately resulting in a loss of immunological tolerance to β cell antigens. Early efforts to link specific risk variants with functional alterations in host immune responses have employed animal models or genotype-selected individuals from clinical bioresource banks. While some notable genotype:phenotype associations have been described, there remains an urgent need to accelerate the discovery of causal variants and elucidate the molecular mechanisms by which susceptible alleles alter immune functions. One significant limitation has been the inability to study human T1D risk loci on an isogenic background. The advent of induced pluripotent stem cells (iPSCs) and genome-editing technologies have made it possible to address a number of these outstanding questions. Specifically, the ability to drive multiple cell fates from iPSC under isogenic conditions now facilitates the analysis of causal variants in multiple cellular lineages. Bioinformatic analyses have revealed that T1D risk genes cluster within a limited number of immune signaling pathways, yet the relevant immune cell subsets and cellular activation states in which candidate risk genes impact cellular activities remain largely unknown. In this review, we summarize the functional impact of several candidate risk variants on host immunity in T1D and present an isogenic disease-in-a-dish model system for interrogating risk variants, with the goal of expediting precision therapeutics in T1D

Evaluation of commonly used ectoderm markers in iPSC trilineage differentiation.

Patient-derived induced pluripotent stem cells (iPSCs) have become a promising resource for exploring genetics of complex diseases, discovering new drugs, and advancing regenerative medicine. Increasingly, laboratories are creating their own banks of iPSCs derived from diverse donors. However, there are not yet standardized guidelines for qualifying these cell lines, i.e., distinguishing between bona fide human iPSCs, somatic cells, and imperfectly reprogrammed cells. Here, we report the establishment of a panel of 30 iPSCs from CD34+ peripheral blood mononuclear cells, of which 10 were further differentiated in vitro into all three germ layers. We characterized these different cell types with commonly used pluripotent and lineage specific markers, and showed that NES, TUBB3, and OTX2 cannot be reliably used as ectoderm differentiation markers. Our work highlights the importance of marker selection in iPSC authentication, and the need for the field to establish definitive standard assays

Enhanced differentiation of human pluripotent stem cells into cardiomyocytes by bacteria-mediated transcription factors delivery

<div><p>Virus-mediated expression of defined transcription factor (TF) genes can effectively induce cellular reprogramming. However, sustained expression of the TFs often hinders pluripotent stem cell (PSC) differentiation into specific cell types, as each TF exerts its effect on PSCs for a defined period of time during differentiation. Here, we applied a bacterial type III secretion system (T3SS)-based protein delivery tool to directly translocate TFs in the form of protein into human PSCs. This transient protein delivery technique showed high delivery efficiency for hPSCs, and it avoids potential genetic alterations caused by the introduction of transgenes. In an established cardiomyocyte <i>de novo</i> differentiation procedure, five transcriptional factors, namely GATA4, MEF2C, TBX5, ESRRG and MESP1 (abbreviated as GMTEM), were translocated at various time points. By detecting the expression of cardiac marker genes (<i>Nkx2</i>.<i>5</i> and <i>cTnT</i>), we found that GMTEM proteins delivered on mesoderm stage of the cardiomyocytes lineage differentiation significantly enhanced both the human ESC and iPSC differentiation into cardiomyocytes, while earlier or later delivery diminished the enhancing effect. Furthermore, all of the five factors were required to enhance the cardiac differentiation. This work provides a virus-free strategy of transient transcription factors delivery for directing human stem cell fate without jeopardizing genome integrity, thus safe for biomedical applications.</p></div

Cytotoxicity assay of <i>P</i>. <i>aeruginosa</i> strain Δ8.

<p>(A) Human ES cell line RUES2 was co-cultured with strain Δ8 or wild-type strain PAK-J for 3 h at indicated MOI (left panel), or for the indicated length of time at MOI of 50 (right panel). After co-culture, the culture supernatants were used for lactate dehydrogenase (LDH) release assay. Data represent means of three biological replicates. Error bars represent SD. *<i>P</i> < 0.05, ^# <i>P</i> < 0.001. (B) Crystal violet staining of the viable hES cells that remain adhered to tissue culture plates after 3 h co-culture with indicated bacterial cells at MOI of 150.</p

GMTEM delivery enhances hiPSC-derived CMs differentiation.

<p>(A) FACS analysis and the percentage of human iPSC derived cTnT⁺ cardiomyocytes. Bar figure show average of three repeats. ^# <i>P</i> < 0.001. (B) Detection of sarcomeric α-actinin expression in the hiPSC-CMs. (C) Relative expression levels of cardiac marker genes in the hiPSC-CMs. *<i>P</i> < 0.05 (n = 3).</p

Schematic representation of T3SS-based transcription factor (TF) delivery.

<p>Schematic representation of T3SS-based transcription factor (TF) delivery.</p