Stem Cell Lines Research Articles

Generation and characterization of two isogenic induced pluripotent stem cell lines from a young female with microcephaly carrying a compound heterozygous mutation in BUB1 gene

Mutations in the Budding uninhibited by benzimidazoles (BUB1) gene were recently associated with neurodevelopmental disorders (Carvalhal et al., 2022). Here, we describe the generation and characterization of two induced pluripotent stem cells (iPSC) clones from a young female with microcephaly. The patient carried two variants in the BUB1 gene (OMIM # 602452), one (c.[2197dupG]; p.[D732fs*11]) paternally inherited and one (c.[2625+1G>A]; p.[V822_L875del] maternally inherited. The generated clones exhibit a normal karyotype (UALGi003-A) and trisomy 8 (UALGi003-B), express pluripotency markers, and differentiate into trilineage cells in vitro. These cell lines can be used to study neurodevelopment and the processes of chromosome segregation.

Neural crest precursors from the skin are the primary source of directly reprogrammed neurons

Direct reprogramming involves the conversion of differentiated cell types without returning to an earlier developmental state. Here, we explore how heterogeneity in developmental lineage and maturity of the starting cell population contributes to direct reprogramming using the conversion of murine fibroblasts into neurons. Our hypothesis is that a single lineage of cells contributes to most reprogramming and that a rare elite precursor with intrinsic bias is the source of reprogrammed neurons. We find that nearly all reprogrammed neurons are derived from the neural crest (NC) lineage. Moreover, when rare proliferating NC precursors are selectively ablated, there is a large reduction in the number of reprogrammed neurons. Previous interpretations of this paradigm are that it demonstrates a cell fate conversion across embryonic germ layers (mesoderm to ectoderm). Our interpretation is that this is actually directed differentiation of a neural lineage stem cell in the skin that has intrinsic bias to produce neuronal progeny.

SNPs in cytochromes P450 catalyzing cholesterol degradation in brain are associated with Parkinson's disease.

Besides being an essential structural component of plasma membranes and the precursor of many functional compounds and signaling molecules, cholesterol was also proposed to play a role in the etiology and/or manifestation of Parkinson's disease (PD). However, so far systematic investigations on the role of cholesterol and its metabolites present in the brain for the etiology of PD are missing. Here, we investigate for the first time the association of PD with SNPs in the genes of four cytochromes P450 (P450), CYP46A1, CYP39A1, CYP27A1 and CYP7B1, which are critical for the degradation of cholesterol in the brain. Analyzing 1,349 individuals from the PPMI data base, we found 24 SNPs in these four genes, which are significantly over- or under-represented in patients suffering from idiopathic PD (IPD). Studying each of the 362 IPD patients individually, we found that most patients (45%) showed only one associated SNP in one of the four P450 genes, while 31% displayed two associated SNPs and 18% three associated SNPs. The occurrence of some associated SNPs is in the same order of magnitude as SNPs in the GBA (beta-glucocerebrosidase) and thus might reflect a genetic predisposition for PD. As all 24 SNPs were located in introns and 3' untranslated regions, we evaluated the prospective regulatory impact of the surrounding genomic regions by using transcriptome and epigenome data from the Foundational Data Initiative for Parkinson Disease (FOUNDIN-PD). FOUNDIN-PD provides gene expression, open chromatin and DNA methylation data in a cohort of 89 induced pluripotent stem cell (iPSC) lines differentiated to dopaminergic (DA) neurons derived from people in the PPMI study. Indeed, two of the 24 SNPs, one in CYP7B1 (rs118111353) and the other one in CYP27A1 (rs74446825), were localized within a region of open chromatin in differentiated neurons. Interestingly, all iPSC lines with open chromatin in rs118111353 showed the reference allele. As all four P450, CYP46A1, CYP39A1, CYP27A1 and CYP7B1, are expressed in dopaminergic neurons, we discuss further functional studies to connect SNPs in regulatory regions with gene expression levels. Finally, potential possibilities for personalized therapeutic treatment of patients with SNPs in the four investigated P450 are discussed.

Phenolic Compounds Isolated from Thlaspi Arvense and Their Effects on Osteoblast Differentiation in the Mouse Mesenchymal Stem Cell Line C3H10T1/2

Phenolic Compounds Isolated from <i>Thlaspi Arvense</i> and Their Effects on Osteoblast Differentiation in the Mouse Mesenchymal Stem Cell Line C3H10T1/2

A high-throughput gene expression analysis software tool for developmental time series and gene signature analysis of human cardiomyocyte differentiation

Publicly available high-throughput gene expression data enable the investigation of biological processes by the scientific community. Although several bioinformatics tools offer methodologies for basic differential gene expression analysis, difficulties arise in the analysis of multiple sample groups comprising a developmental time series, especially when the identification and classification of unique gene expression patterns is the primary goal of the study. Data analysis using these tools requires programming experience, which limits the accessibility of these tools to the broader community. To streamline developmental time-series investigations, we created the Developmental Gene Expression Analysis (devGEA) tool. This environment can be implemented locally or via web browsers to expedite differential gene expression analysis. This tool provides gene signature determination methods that can classify differentially expressed genes based on their correlation with gene expression patterns. devGEA was used to characterize cardiac development gene expression signatures from high-throughput RNA-seq datasets profiling small-molecule directed cardiomyocyte differentiation of human pluripotent stem cell lines (hiPSCs). After pre-processing, discrete gene expression criteria-based expected changes were used to classify the genes into developmental signatures. Several cardiomyocyte differentiation markers and candidate cardiac genes representing different developmental signatures were experimentally validated using the GIBCOTM hiPSC line. This method was then compared to a gene signature correlation approach that classified expressed genes based on their degree of similarity with key cardiac developmental signatures representing the stages of cardiomyocyte differentiation. Therefore, devGEA provides a robust workflow for investigator-driven analysis of developmental time series, allowing for the identification of differentially expressed genes and gene signatures for extensive experimental investigation. We also introduced a method for classifying genes by their correlation with genes or developmental patterns of interest. Our correlation-based method takes advantage of a priori knowledge of an experiment and is conceptually simpler than unsupervised clustering approaches.

Optimization of Exon-Skipping Riboswitches and Their Applications to Control Mammalian Cell Fate.

Mammalian riboswitches that can regulate transgene expression via RNA-small molecule interaction have promising applications in medicine and biotechnology, as they involve no protein factors that can induce immunogenic reactions and are not dependent on specially engineered promoters. However, the lack of cell-permeable and low-toxicity small molecules and cognate aptamers that can be exploited as riboswitches and the modest switching performance of mammalian riboswitches have limited their applications. In this study, we systematically optimized the design of a riboswitch that regulates exon skipping via an RNA aptamer that binds ASP2905. We examined two design strategies to modulate the stability of the aptamer base stem that blocks the 5' splice site to fine-tune the riboswitch characteristics. Furthermore, an optimized riboswitch was used to generate a mouse embryonic stem cell line that can be chemically induced to differentiate into myogenic cells by activating Myod1 expression and a human embryonic kidney cell line that can be induced to trigger apoptosis by activating BAX expression. The results demonstrate the tight chemical regulation of transgenes in mammalian cells to control their phenotype without exogenous protein factors.

Elavl1 is dispensable for appendicular skeletal development.

Elavl1/HuR is a RNA binding protein implicated in multiple developmental processes with pleiotropic roles in RNA life cycle. Loss of Elavl1 is incompatible with life with early embryonic loss of Elavl1 in epiblast cells being lethal with defects in placental branching and embryonic tissue growth. Postnatal global deletion of Elavl1/HuR results in lethality with atrophy in multiple tissues mainly due to loss of progenitor cells. However, roles of Elavl1 specifically during embryonic limb development is not well understood. Here we report that deletion of Elavl1 in limb bud mesenchyme in mouse did not reveal any abnormalities during embryonic development with normal development in pre- and postnatal limb skeleton. Analyses of skeletal patterning, morphogenesis and skeletal maturation including skeletal elements in stylopod, zeugopod and autopod during development did not reveal any significant differences between long bones from control and Elavl1 conditional knockout animals. Our study indicates differential dependency and susceptibility to loss of Elavl1 in different stem cell lineages with its functions being dispensable during limb development.

Generation of ID1/3 knockout human embryonic stem cell lines (WAe009-A-2A and WAe009-A-2B) derived from H9 using CRISPR/Cas9

ID1 and ID3 are the members of the Inhibitor of DNA Binding (ID) protein family, which negatively regulates the basic helix-loop-helix (bHLH) transcription factors by forming heterodimers, are involved in neurodevelopment, cardiovascular development, and tumor metastasis. We created twoID1/3knockout cell lines from a human embryonic stem cell (hESC) line (H9) by CRISPR/Cas9-mediated gene targeting. These cell lines maintain stem cell morphology, a normal karyotype, and the expression of pluripotent marker genes. Additionally, they retain their in vivo differentiation potential. Thecell lines are valuable tools for studying the roles of ID1/3 in neurodevelopment and cardiovascular diseases.

Modulating DNA Polα Enhances Cell Reprogramming Across Species.

As a fundamental biological process, DNA replication ensures the accurate copying of genetic information. However, the impact of this process on cellular plasticity in multicellular organisms remains elusive. Here, we find that reducing the level or activity of a replication component, DNA Polymerase α (Polα), facilitates cell reprogramming in diverse stem cell systems across species. In Drosophila male and female germline stem cell lineages, reducing Polα levels using heterozygotes significantly enhances fertility of both sexes, promoting reproductivity during aging without compromising their longevity. Consistently, in C. elegans the pola heterozygous hermaphrodites exhibit increased fertility without a reduction in lifespan, suggesting that this phenomenon is conserved. Moreover, in male germline and female intestinal stem cell lineages of Drosophila, polα heterozygotes exhibit increased resistance to tissue damage caused by genetic ablation or pathogen infection, leading to enhanced regeneration and improved survival during post-injury recovery, respectively. Additionally, fine tuning of an inhibitor to modulate Polα activity significantly enhances the efficiency of reprogramming human embryonic fibroblasts into induced pluripotent cells. Together, these findings unveil novel roles of a DNA replication component in regulating cellular reprogramming potential, and thus hold promise for promoting tissue health, facilitating post-injury rehabilitation, and enhancing healthspan.

Generation of an induced pluripotent stem cell line from a type 1 neurofibromatosis patient with NF1 mutation

A human induced pluripotent stem cell (iPSC) line was generated from patient with type 1 neurofibromatosis (NF-1), carrying heterozygous mutation in NF1 gene. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using non-integrating delivery of KFL4, OCT4, SOX2, BCL-XL and c-MYC. The iPSC line expresses pluripotency markers, displays a normal karyotype, and is able to differentiate into three germ layers in vitro. This iPSC line represents a valuable cell model for NF1 in humans.

Generation of footprint-free human induced pluripotent stem cell line (SCIKFi001-B) from cGMP grade umbilical cord-derived mesenchymal stem cells (UC-MSCs) using episomal-plasmid based reprogramming approach

UCMSCs were reprogrammed to iPSCs using Yamanaka factor bearing episomal plasmids. SCIKFi001-B exhibited pluripotency, had typical iPSC morphology and didn’t retain any residual episomal plasmid. Although karyotyping showed chromosomal translocation, this abnormality seemed to have little impact on the functionality of SCIKFi001-B since it retained its ability to differentiate to three-germ layer. While karyotypic abnormality might negate use in therapeutic and clinical settings, this line remained a valuable educational tool for iPS cell culture techniques. Finally, our study highlighted the importance of routine karyotyping on iPSC lines as abnormal karyotypes oftentimes bear no discernible effect on cell morphology nor functionality.

Strategic naturalizing in the Anthropocene: Managing cells, bodies and ecosystems

Discussions on the Anthropocene have called for increased attention to how the effects of human actions on the planet are accounted for. While much of this debate remains at a theoretical level, more studies on situated Anthropocene realities have been called for. Contributing to the latter, this article explores how experimental and clinical interventions are being accounted for in life science laboratories. Drawing on three research cases, genetically modified mosquitoes, laboratory-grown stem cell lines and assisted reproduction in the IVF clinic, the article traces how ‘strategic naturalizing’ is conducted to make sense of and justify human interventions on cells, bodies and ecosystems. Strategic naturalizing in laboratory work is situated, fluid, and also implicates the presence of the social scientist. Although naturalizing biotechnologies is not a new phenomenon, according to our observations scientists increasingly draw on notions of a planet profoundly transformed by human intervention as they conceptualize their own accountability. Consequently, we propose that strategic naturalizing is emerging as an elemental logic in the patchy local enactments of the Anthropocene and its concerns. In the context of experimental and clinical laboratory work, it is a key element in enacting good science and orienting the work towards making biotechnology acceptable in wider society. In studying this, the social scientist also is implicated in situated local enactments of the Anthropocene.

Six induced pluripotent stem cell lines from fibroblasts of individuals with CLN3-related conditions

Primary fibroblasts from six individuals with CLN3-related conditions were used to generate induced pluripotent stem cell (iPSC) lines CHDTRi001-B, CHDTRi002-B, CHDTRi003-A, CHDTRi004-B, CHDTRi005-A, and CHDTRi006-E through the expression of four reprogramming factors: human OCT3/4, KLF4, SOX2, and c-MYC. The iPSC lines were characterized to confirm their pluripotency via immunocytochemistry, flow cytometry, and teratoma formation. Genomic stability, cell line identity, and CLN3 genotype were confirmed. These iPSC lines may be used as participant-derived experimental models for further investigation of CLN3, a rare, fatal, pediatric, blindness and neurodegenerative lysosomal disorder with no cure.

Can the male germline offer insight into mammalian brain size expansion?

Recent advances in single-cell transcriptomic data have greatly expanded our understanding of both spermatogenesis and the molecular mechanisms of male infertility. However, this growing wealth of data could also shed light on a seemingly unrelated biological problem: the genetic basis of mammalian brain size expansion throughout evolution. It is now increasingly recognized that the testis and brain share many cellular and molecular similarities including pivotal roles for the RAS/MAPK and PI3K/AKT/mTOR pathways, mutations in which are known to have a pronounced impact on cell proliferation. Most notably, in the stem cell lineages of both organs, new mutations have been shown to increase cellular output over time. These include 'selfish' mutations in spermatogonial stem cells, which disproportionately increase the proportion of mutant sperm, and-to draw a parallel-human-specific mutations in neural stem cells which, by increasing the number of neurons, have been implicated in neocortical expansion. Here we speculate that the origin for many 'expansion'-associated mutations is the male germline and that as such, a deeper understanding of the mechanisms controlling testicular turnover may yield fresh insight into the biology and evolution of the brain.

Human induced pluripotent stem cell line (FDHSi005-A) derived from a patient with a deep intronic variant in the GNE gene

GlcNAc2-epimerase myopathy is a rare autosomal recessive myopathy characterized by distal involvement in the lower extremities. Our study reprogrammed human-induced pluripotent stem cells from peripheral blood mononuclear cells of a patient with GNE gene deep intronic variant c.862 + 870C>T and c.478C>T compound heterozygous mutations that co-segregated with the disease. The generated iPSCs express pluripotent cell markers with no mycoplasma contamination. Additionally, these iPSCs demonstrated pluripotency, the capacity to differentiate into the three germ layers, and maintained normal karyotypes. Importantly, we identified that these iPSCs possess the same specific mutations as the patient, making them a robust model for studying GNE myopathy and developing potential therapeutic interventions.

Stem Cell Treatment for Dilated Cardiomyopathy: A Review of Recent Scientific Advances

Cardiomyopathy, a disorder of mechanical and electrical cardiac action, is a significant cause of morbidity and mortality in the United States and is seen in both adult and pediatric populations. Dilated cardiomyopathy, a subtype involving pathological enlargement of the heart, in particular causes significant reduction in quality of life and is a significant risk factor for heart failure development. Traditional treatment for dilated cardiomyopathy largely focuses on symptomatic relief and delayed progression of disease but does not offer a cure or reversal of disease process. In the past few decades, stem cell therapy has become a promising therapeutic because these cells can differentiate into human cardiomyocytes and offer a potential restoration of normal cardiomyocyte function. This review highlights 3 methodological derivations of cardiomyocytes: bone marrow–derived hematopoietic stem cells, mesenchymal stem cells, and adipose-derived stem cells. While most of the current research has focused on hematopoietic stem cells for dilated cardiomyopathy, further research is needed to elucidate which stem cell line is superior and most effective for disease treatment and reversal. This review highlights future directions of this line of research, such as direct comparison between cell types, analysis of the long-term effects of stem cell treatment, increased sample sizes, and potential use of this treatment in the pediatric and adult populations.

Generation of human induced pluripotent stem cell lines from a subject with UBAP1L-associated retinal dystrophy and CRISPR/cas9-corrected isogenic iPSC lines

A Human induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of a patient affected with an autosomal recessive retinal dystrophy carrying the homozygous c.910-7G>A variant in UBAP1L. Three isogenic control iPSC lines derived from this affected subject line were created using CRISPR/Cas9 engineering. All iPSC lines expressing the pluripotency markers, were able to differentiate into the three germ layers, and exhibit a normal karyotype. These cellular models will provide a powerful tool to study disease mechanisms associated with the recently reported UBAP1L- associated retinal dystrophy and better understand the role of the protein in retinal physiology.

Generation of a human induced pluripotent stem cell line (FDIBSi001-A) from a patient with ADNP syndrome carrying ADNP mutation (c. 2059 T>C)

ADNP syndrome is a neurodevelopmental disorder characterized by autism, intellectual disability, and other physical and behavioral health manifestations. Mutations in ADNP gene is responsible for ADNP syndrome. A human iPSC line with a de novo heterozygous ADNP mutation (ADNP c. 2059 T>C) was generated from peripheral blood mononuclear cells of a patient with ADNP syndrome. This iPSC line showed typical human embryonic stem cell-like morphology, normal karyotype, pluripotency, and ability to differentiate into three germ layers. This iPSC line provides a useful resource to study the pathogenesis and drug screening of ADNP syndrome.

Generation of a fluorescent mNeonGreen insulin reporter line in the H1 (WA01) hESC background

Over the past decade, the use of human stem cell-derived β cells (SC-β cells) to model pancreatic β cell development, function and disease has become increasingly common. Though protocols are rapidly improving, current directed differentiation strategies do not yield a pure population of insulin-positive SC-β cells in vitro. Therefore, it is experimentally advantageous to have reporter lines that allow for live sorting of insulin-positive populations. To aid in these studies, we have knocked mNeonGreen fluorescent protein into the endogenous insulin locus of the commonly used H1 (WA01) human embryonic stem cell line.

Generation and genetic repair of two human induced pluripotent stem cell lines from patients with Epidermolysis Bullosa simplex associated with a heterozygous mutation in the translation initiation codon of KLHL24

Fibroblasts from two patients carrying a distinct heterozygous mutation in the KLHL24 gene (c.1 A>G and c.2T>C) were reprogrammed to obtain hiPSC lines. Non-integrating Sendai virus and CRISPR-Cas9 editing were respectively used to deliver the reprogramming factors and repair the mutation in the patient-hiPSCs to obtain isogenic control pairs. No off-target nuclease activity was detected with the top-predicted sites. Patient and isogenic hiPSCs displayed typical morphology, expressed markers of the undifferentiated state, were able to differentiate into the three germ layers and had normal karyotypes. These isogenic pairs will expand the panel of hiPSC lines to model KLHL24-associated conditions.