The distinct effects of P18 overexpression on different stages of hematopoiesis involve TGF-β and NF-κB signaling

Deficiency of P18 can significantly improve the self-renewal potential of hematopoietic stem cells (HSC) and the success of long-term engraftment. However, the effects of P18 overexpression, which is involved in the inhibitory effects of RUNX1b at the early stage of hematopoiesis, have not been examined in detail. In this study, we established inducible P18/hESC lines and monitored the effects of P18 overexpression on hematopoietic differentiation. Induction of P18 from day 0 (D0) dramatically decreased production of CD34highCD43− cells and derivative populations, but not that of CD34lowCD43− cells, changed the cell cycle status and apoptosis of KDR+ cells and downregulated the key hematopoietic genes at D4, which might cause the severe blockage of hematopoietic differentiation at the early stage. By contrast, induction of P18 from D10 dramatically increased production of classic hematopoietic populations and changed the cell cycle status and apoptosis of CD45+ cells at D14. These effects can be counteracted by inhibition of TGF-β or NF-κB signaling respectively. This is the first evidence that P18 promotes hematopoiesis, a rare property among cyclin-dependent kinase inhibitors (CKIs).

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

Although many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

A quantitative comparison of human embryonic and induced stem cell proteomes

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. However, a clear overview of their differences at the protein level is still incomplete. In this study we characterise the proteomes of hiPSC and hESC lines, where we find that they express a similar set of proteins but show consistent quantitative differences that can be masked by the normalisation methods. hiPSCs have a higher protein content, with over 1,500 proteins showing over two-fold increased expression. They also display proteomic differences in their mitochondria, with increased expression of mitochondrial transporters and metabolic proteins as well as mitochondrial translation machinery. The hiPSCs also show higher expression of important amino acid transporters, secreted proteins, and growth factors with potential to affect neighbouring cells, coupled with a systematic reduction in the expression levels of H1 histone variants. We conclude that despite hiPSCs and hESCs being highly similar cell types, they show important differences in protein expression that may be relevant for their use in clinical research.

MFN2 Deficiency Impairs Mitochondrial Transport and Downregulates Motor Protein Expression in Human Spinal Motor Neurons

Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.

Changes in Methylation Patterns of Tumor Suppressor Genes during Extended Human Embryonic Stem Cell Cultures

While studies on embryonic stem cells have been actively conducted, little is known about the epigenetic mechanisms in human embryonic stem cells (hESCs) in extended culture systems. Here, we investigated whether CpG island (CGI) methylation patterns of 24 tumor suppressor genes could be maintained during extended hESC cultures. In total, 10 hESC lines were analyzed. For each cell line, genomic DNA was extracted from early and late passages of cell cultures. CGI methylation levels of 24 tumor suppressor genes were analyzed using methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA), pyrosequencing, and real-time polymerase chain reaction (PCR). Different CGI methylation patterns of CASP8, FHIT, and CHFR genes were identified in between early and late passages in some hESC lines. CGI methylation levels of CASP8 significantly increased at late passage in CHA-36, CHA-40, and CHA-42 cell lines compared to those at early passage. The CGI methylation of the FHIT gene was higher at late passage than at early passage in CHA-15, CHA-31, CHA-32, and iPS (FS)-1 cell lines but decreased at the late passage in CHA-20 and H1 cell lines. Different CGI methylation patterns were detected for the CHFR gene only in iPS (FS)-1, and the level significantly increased at late passage. Thus, our findings show that CGI methylation patterns could be altered during prolonged ESC cultures and examining these epigenetic changes is important to assess the maintenance, differentiation, and clinical usage of stem cells.

A Benchmark Side-by-Side Comparison of Two Well-Established Protocols for in vitro Hematopoietic Differentiation From Human Pluripotent Stem Cells

The generation of transplantable hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) remains challenging. Current differentiation protocols from hPSCs generate mostly hematopoietic progenitors of the primitive HSC-independent program, and it remains unclear what is the best combination of cytokines and hematopoietic growth factors (HGFs) for obtaining functional hematopoietic cells in vitro. Here, we have used the AND1 and H9 hESC lines and the H9:dual-reporter RUNX1C-GFP-SOX17-Cherry to compare the hematopoietic differentiation in vitro based on the treatment of embryoid bodies (EBs) with the ventral mesoderm inducer BMP4 plus HGFs in the absence (protocol 1) or presence (protocol 2) of stage-specific activation of Wnt/β-catenin and inhibition of Activin/Nodal. Despite a slight trend in favor of protocol 1, no statistically significant differences were observed between protocols at any time point analyzed throughout EB development regarding the frequency of hemogenic endothelial (HE) precursors; CD43+ CD45−, CD45+, and CD45 + CD34 + hematopoietic derivatives; or the output of clonogenic progenitors. Similarly, the kinetics of emergence throughout EB development of both SOX17 + HE and RUNX1C + definitive hematopoiesis was very similar for both protocols. The expression of the early master mesendodermal transcription factors Brachyury, MIXL1, and KDR revealed similar gene expression kinetics prior to the emergence of RUNX1C + definitive hematopoiesis for both protocols. Collectively, the simpler protocol 1 is, at least, as efficient as protocol 2, suggesting that supplementation with additional morphogens/HGFs and modulation of Activin/Nodal and Wnt/β-catenin pathways seem dispensable for in vitro hematopoietic differentiation of hPSCs.

Heterozygous APC germline mutations impart predisposition to colorectal cancer

Familial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how different APC germline mutations predict disease severity. Three FAP-human embryonic stem cell lines (FAP1/2/3-hESC lines) carrying germline mutations at different locations of the APC gene, and two control hESC lines free of the APC mutation, were differentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP-hESCs carrying a complete loss-of-function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP-hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst-like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical differentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non-pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non-FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufficient to initiate early molecular tumorigenic activity. In addition, the results hint that patient-specific hESC-derived colon organoids can probably predict disease severity among FAP patients.

Genome-wide CRISPR interference screen identifies long non-coding RNA loci required for differentiation and pluripotency

ABSTRACTAlthough many long non-coding RNAs (lncRNAs) exhibit lineage-specific expression, the vast majority remain functionally uncharacterized in the context of development. Here, we report the first described human embryonic stem cell (hESC) lines to repress (CRISPRi) or activate (CRISPRa) transcription during differentiation into all three germ layers, facilitating the modulation of lncRNA expression during early development. We performed an unbiased, genome-wide CRISPRi screen targeting thousands of lncRNA loci expressed during endoderm differentiation. While dozens of lncRNA loci were required for proper differentiation, most differentially expressed lncRNAs were not, supporting the necessity for functional screening instead of relying solely on gene expression analyses. In parallel, we developed a clustering approach to infer mechanisms of action of lncRNA hits based on a variety of genomic features. We subsequently identified and validated FOXD3-AS1 as a functional lncRNA essential for pluripotency and differentiation. Taken together, the cell lines and methodology described herein can be adapted to discover and characterize novel regulators of differentiation into any lineage.

Biological insights from the whole genome analysis of human embryonic stem cells

ABSTRACTThere has not yet been a systematic analysis of hESC whole genomes at a single nucleotide resolution. We therefore performed whole genome sequencing (WGS) of 143 hESC lines and annotated their single nucleotide and structural genetic variants. We found that while a substantial fraction of hESC lines contained large deleterious structural variants, finer scale structural and single nucleotide variants (SNVs) that are ascertainable only through WGS analyses were present in hESCs genomes and human blood-derived genomes at similar frequencies. However, WGS did identify SNVs associated with cancer or other diseases that will likely alter cellular phenotypes and may compromise the safety of hESC-derived cellular products transplanted into humans. As a resource to enable reproducible hESC research and safer translation, we provide a user-friendly WGS data portal and a data-driven scheme for cell line maintenance and selection.GRAPHICAL ABSTRACTIN BRIEFMerkle and Ghosh et al. describe insights from the whole genome sequences of commonly used human embryonic stem cell (hESC) lines. Analyses of these sequences show that while hESC genomes had more large structural variants than humans do from genetic inheritance, hESCs did not have an observable excess of finer-scale variants. However, many hESC lines contained rare loss-of-function variants and combinations of common variants that may profoundly shape their biological phenotypes. Thus, genome sequencing data can be valuable to those selecting cell lines for a given biological or clinical application, and the sequences and analysis reported here should facilitate such choices.HIGHLIGHTSOne third of hESCs we analysed are siblings, and almost all are of European ancestryLarge structural variants are common in hESCs, but finer-scale variation is similar to that human populationsMany strong-effect loss-of-function mutations and cancer-associated mutations are present in specific hESC linesWe provide user-friendly resources for rational hESC line selection based on genome sequence