Epigenetic repression of CHCHD2 enhances survival from single cell dissociation through attenuated Rho A kinase activity.

During in vitro culture, human pluripotent stem cells (hPSCs) often acquire survival advantages characterized by decreased susceptibility to mitochondrial cell death, known as "culture adaptation." This adaptation is associated with genetic and epigenetic abnormalities, including TP53 mutations, copy number variations, trisomy, and methylation changes. Understanding the molecular mechanisms underlying this acquired survival advantage is crucial for safe hPSC-based cell therapies. Through transcriptome and methylome analysis, we discovered that the epigenetic repression of CHCHD2, a mitochondrial protein, is a common occurrence during in vitro culture using enzymatic dissociation. We confirmed this finding through genetic perturbation and reconstitution experiments in normal human embryonic stem cells (hESCs). Loss of CHCHD2 expression conferred resistance to single cell dissociation-induced cell death, a common stress encountered during in vitro culture. Importantly, we found that the downregulation of CHCHD2 significantly attenuates the activity of Rho-associated protein kinase (ROCK), which is responsible for inducing single cell death in hESCs. This suggests that hESCs may survive routine enzyme-based cell dissociation by downregulating CHCHD2 and thereby attenuating ROCK activity. These findings provide insights into the mechanisms by which hPSCs acquire survival advantages and adapt to in vitro culture conditions.

Rnf220 cooperates with Zc4h2 to specify spinal progenitor domains.

During early embryonic development of the spinal cord, graded sonic hedgehog signaling establishes distinct ventral progenitor domains by regulating the spatiotemporal expression of fate-specifying transcription factors. However, regulation of their protein stability remains incompletely understood. Here, we show that RNF220, an E3 ubiquitin ligase, plays crucial roles in the generation of the ventral progenitor domains, which produce ventral interneurons and motor neurons, by targeting key transcription factors including Dbx1/2 and Nkx2.2 for degradation. Surprisingly, RNF220 interacts with, and is co-expressed with, a zinc-finger protein ZC4H2, and they cooperate to degrade Dbx1/2 and Nkx2.2. RNF220-null mice show widespread alterations of ventral progenitor domains, including the loss of the p2 domain that produces V2 interneurons. Knockdown of RNF220 and ZC4H2 in the chick spinal cord downregulates expression of the V2 interneuronal marker Chx10. Co-expression of RNF220 and ZC4H2 further promotes the ability of Nkx6.1 to induce ectopic Chx10+ V2 interneurons. Our results uncover a novel regulatory pathway in establishing distinct progenitor domains through modulating the protein stability of transcription factors. Our results provide insights into the molecular mechanism by which ZC4H2 mutations lead to human syndromes characterized by delayed motor development.

MutSα and MutSß as size-dependent cellular determinants for prime editing in human embryonic stem cells.

Precise genome editing in human pluripotent stem cells (hPSCs) has potential applications in isogenic disease modeling and ex vivo stem cell therapy, necessitating diverse genome editing tools. However, unlike differentiated somatic cells, hPSCs have unique cellular properties that maintain genome integrity, which largely determine the overall efficiency of an editing tool. Considering the high demand for prime editors (PEs), it is imperative to characterize the key molecular determinants of PE outcomes in hPSCs. Through homozygous knockout (KO) of MMR pathway key proteins MSH2, MSH3, and MSH6, we reveal that MutSα and MutSß determine PE efficiency in an editing size-dependent manner. Notably, MSH2 perturbation disrupted both MutSα and MutSß complexes, dramatically escalating PE efficiency from base mispair to 10 bases, up to 50 folds. Similarly, impaired MutSα by MSH6 KO improved editing efficiency from single to three base pairs, while defective MutSß by MSH3 KO heightened efficiency from three to 10 base pairs. Thus, the size-dependent effect of MutSα and MutSß on prime editing implies that MMR is a vital PE efficiency determinant in hPSCs and highlights the distinct roles of MutSα and MutSß in its outcome.

Partial in vivo reprogramming enables injury-free intestinal regeneration via autonomous Ptgs1 induction.

Tissue regeneration after injury involves the dedifferentiation of somatic cells, a natural adaptive reprogramming that leads to the emergence of injury-responsive cells with fetal-like characteristics. However, there is no direct evidence that adaptive reprogramming involves a shared molecular mechanism with direct cellular reprogramming. Here, we induced dedifferentiation of intestinal epithelial cells using OSKM (Oct4, Sox2, Klf4, and c-Myc) in vivo. The OSKM-induced forced dedifferentiation showed similar molecular features of intestinal regeneration, including a transition from homeostatic cell types to injury-responsive-like cell types. These injury-responsive-like cells, sharing gene signatures of revival stem cells and atrophy-induced villus epithelial cells, actively assisted tissue regeneration following damage. In contrast to normal intestinal regeneration involving Ptgs2 induction, the OSKM promotes autonomous production of prostaglandin E2 via epithelial Ptgs1 expression. These results indicate prostaglandin synthesis is a common mechanism for intestinal regeneration but involves a different enzyme when partial reprogramming is applied to the intestinal epithelium.

Fat3 regulates neural progenitor cells by promoting Yap activity during spinal cord development.

Early embryonic development of the spinal cord requires tight coordination between proliferation of neural progenitors and their differentiation into distinct neuronal cell types to establish intricate neuronal circuits. The Hippo pathway is one of the well-known regulators to control cell proliferation and govern neural progenitor cell number, in which the downstream effector Yes-associated protein (Yap) promotes cell cycle progression. Here we show that an atypical cadherin Fat3, expressed highly in the neural tube, plays a critical role in maintaining proper number of proliferating progenitors. Knockdown of Fat3 in chick neural tube down-regulates expression of the proliferation markers but rather induces the expression of neural markers in the ventricular zone. We further show that deletion of Fat3 gene in mouse neural tube depletes neural progenitors, accompanied by neuronal gene expression in the ventral ventricular zone of the spinal cord. Finally, we found that Fat3 regulates the phosphorylation level of Lats1/2, the upstream kinase of Yap, resulting in dephosphorylation and stabilization of Yap, suggesting Yap as a key downstream effector of Fat3. Our study uncovers another layer of regulatory mechanisms in controlling the activity of Hippo signaling pathway to regulate the size of neural progenitor pools in the developing spinal cord.

High expression of uracil DNA glycosylase determines C to T substitution in human pluripotent stem cells.

Precise genome editing of human pluripotent stem cells (hPSCs) is crucial not only for basic science but also for biomedical applications such as ex vivo stem cell therapy and genetic disease modeling. However, hPSCs have unique cellular properties compared to somatic cells. For instance, hPSCs are extremely susceptible to DNA damage, and therefore Cas9-mediated DNA double-strand breaks (DSB) induce p53-dependent cell death, resulting in low Cas9 editing efficiency. Unlike Cas9 nucleases, base editors including cytosine base editor (CBE) and adenine base editor (ABE) can efficiently substitute single nucleotides without generating DSBs at target sites. Here, we found that the editing efficiency of CBE was significantly lower than that of ABE in human embryonic stem cells (hESCs), which are associated with high expression of DNA glycosylases, the key component of the base excision repair pathway. Sequential depletion of DNA glycosylases revealed that high expression of uracil DNA glycosylase (UNG) not only resulted in low editing efficiency but also affected CBE product purity (i.e., C to T) in hESCs. Therefore, additional suppression of UNG via transient knockdown would also improve C to T base substitutions in hESCs. These data suggest that the unique cellular characteristics of hPSCs could determine the efficiency of precise genome editing.

Multiple isogenic GNE-myopathy modeling with mutation specific phenotypes from human pluripotent stem cells by base editors.

Despite the great potential of disease modeling using human pluripotent stem cells (hPSCs) derived from patients with mutations, lack of an appropriate isogenic control hinders a precise phenotypic comparison due to the bias arising from the dissimilar genetic backgrounds between the control and diseased hPSCs. Herein, we took advantage of currently available base editors (BEs) to epitomize the isogenic disease model from hPSCs. Using this method, we established multiple isogenic GNE myopathy disease models that harbor point mutations on the GNE gene, including four different mutations found in GNE myopathy patients. Four different mutations in the epimerase or kinase domains of GNE revealed mutation-specific hyposialylation and hyposialylation dependent gene signature, which was closely correlated to pathological clinical phenotypes. GNE protein structure modeling based on the mutations, addressed these mutation-specific hyposialylation patterns. Furthermore, treatment with a drug candidate currently under clinical trials showed a mutation-specific drug response in GNE myopathy disease models. These data suggest that derivation of multiple isogenic disease models from hPSCs by using genome editing can enable translationally relevant studies on the pathophysiology of GNE myopathy and drug responses.

Luteolin Induces Selective Cell Death of Human Pluripotent Stem Cells.

Despite recent advances in clinical stem cell therapy applications based on human pluripotent stem cells (hPSCs), potential teratoma formation due to the presence of residual undifferentiated hPSCs remains a serious risk factor that challenges widespread clinical application. To overcome this risk, a variety of approaches have been developed to eliminate the remaining undifferentiated hPSCs via selective cell death induction. Our study seeks to identify natural flavonoids that are more potent than quercetin (QC), to selectively induce hPSC death. Upon screening in-house flavonoids, luteolin (LUT) is found to be more potent than QC to eliminate hPSCs in a p53-dependent manner, but not hPSC-derived smooth muscle cells or perivascular progenitor cells. Particularly, treating human embryonic stem cell (hESC)-derived cardiomyocytes with LUT efficiently eliminates the residual hESCs and only results in marginal effects on cardiomyocyte (CM) functions, as determined by calcium influx. Considering the technical limitations of isolating CMs due to a lack of exclusive surface markers at the end of differentiation, LUT treatment is a promising approach to minimize teratoma formation risk.

Safe scarless cassette-free selection of genome-edited human pluripotent stem cells using temporary drug resistance.

An efficient gene-editing technique for use in human pluripotent stem cells (hPSCs) has great potential value in regenerative medicine, as well as in drug discovery based on isogenic human disease models. However, the extremely low efficiency of gene editing in hPSCs remains as a major technical hurdle. Previously, we demonstrated that YM155, a survivin inhibitor developed as an anti-cancer drug, induces highly selective cell death in undifferentiated hPSCs. In this study, we demonstrated that the high cytotoxicity of YM155 in hPSCs, which is mediated by selective cellular uptake of the drug, is due to the high expression of SLC35F2 in these cells. Knockout of SLC35F2 with CRISPR-Cas9, or depletion with siRNAs, made the hPSCs highly resistant to YM155. Simultaneous editing of a gene of interest and transient knockdown of SLC35F2 following YM155 treatment enabled the survival of genome-edited hPSCs as a result of temporary YM155 resistance, thereby achieving an enriched selection of clonal populations with gene knockout or knock-in. This precise and efficient genome editing approach took as little as 3 weeks and required no cell sorting or the introduction of additional genes, to be a more feasible approach for gene editing in hPSCs due to its simplicity.