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C-C chemokine receptor type 5 (CCR5) is a major co-receptor for the entry of human immunodeficiency virus type-1 (HIV-1) into target cells. Human hematopoietic stem cells (hHSCs) with naturally occurring CCR5 deletions (Δ32) or artificially disrupted CCR5 have shown potential for curing acquired immunodeficiency syndrome (AIDS). However,Δ32 donors are scarce,heterologous bone marrow transplantation is not exempt of risks,and genetic engineering of autologous hHSCs is not trivial. Here,we have disrupted the CCR5 locus of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) using specific zinc finger nucleases (ZFNs) combined with homologous recombination. The modified hESCs and hiPSCs retained pluripotent characteristics and could be differentiated in vitro into CD34(+) cells that formed all types of hematopoietic colonies. Our results suggest the potential of using patient-specific hHSCs derived from ZFN-modified hiPSCs for treating AIDS. View Publication

Significant efforts have been invested into the differentiation of stem cells into functional hepatocyte-like cells that can be used for cell therapy,disease modeling and drug screening. Most of these efforts have been concentrated on the use of growth factors to recapitulate developmental signals under in vitro conditions. Using small molecules instead of growth factors would provide an attractive alternative since small molecules are cell-permeable and cheaper than growth factors. We have developed a protocol for the differentiation of human embryonic stem cells into hepatocyte-like cells using a predominantly small molecule-based approach (SM-Hep). This 3 step differentiation strategy involves the use of optimized concentrations of LY294002 and bromo-indirubin-3'-oxime (BIO) for the generation of definitive endoderm; sodium butyrate and dimethyl sulfoxide (DMSO) for the generation of hepatoblasts and SB431542 for differentiation into hepatocyte-like cells. Activin A is the only growth factor required in this protocol. Our results showed that SM-Hep were morphologically and functionally similar or better compared to the hepatocytes derived from the growth-factor induced differentiation (GF-Hep) in terms of expression of hepatic markers,urea and albumin production and cytochrome P450 (CYP1A2 and CYP3A4) activities. Cell viability assays following treatment with paradigm hepatotoxicants Acetaminophen,Chlorpromazine,Diclofenac,Digoxin,Quinidine and Troglitazone showed that their sensitivity to these drugs was similar to human primary hepatocytes (PHHs). Using SM-Hep would result in 67% and 81% cost reduction compared to GF-Hep and PHHs respectively. Therefore,SM-Hep can serve as a robust and cost effective replacement for PHHs for drug screening and development. View Publication

To investigate the influence of antibody structure and specificity on antibody efficacy against Streptococcus pneumoniae,human monospecific antibodies (MAbs) to serotype 3 pneumococcal capsular polysaccharide (PPS-3) were generated from transgenic mice reconstituted with human immunoglobulin loci (XenoMouse mice) vaccinated with a PPS-3-tetanus toxoid conjugate and their molecular genetic structures,epitope specificities,and protective efficacies in normal and complement-deficient mice were determined. Nucleic acid sequence analysis of three MAbs (A7,1A2,and 7C5) revealed that they use two different V(H)3 genes (A7 and 1A2 both use V3-15) and three different V(kappa) gene segments. The MAbs were found to have similar affinities for PPS-3 but different epitope specificities and CDR3 regions. Both A7 and 7C5 had a lysine at the V(H)-D junction,whereas 1A2 had a threonine. Challenge experiments with serotype 3 S. pneumoniae in BALB/c mice revealed that both 10- and 1- micro g doses of A7 and 7C5 were protective,while only a 10- micro g dose of 1A2 was protective. Both A7 and 7C5 were also protective in mice lacking either an intact alternative (FB(-/-)) or classical (C4(-/-)) complement pathway,but 1A2 was not protective in either strain. Our data suggest that PPS-3 consists of epitopes that can elicit both highly protective and less protective antibodies and that the superior efficacies of certain antibodies may be a function of their structures and/or specificities. Further investigation of relationships between structure,specificity,and efficacy for defined MAbs to PPS may identify antibody features that might be useful surrogates for antibody (and vaccine) efficacy. View Publication

Cell type-specific surface markers offer a powerful tool for purifying defined cell types for restorative therapies and drug screenings. Midbrain dopaminergic neurons (mesDA) are the nerve cells preferentially lost in the brains of Parkinson's disease patients. Clinical trials of transplantation of fetal neural precursors suggest that cell therapy may offer a cure for this devastating neurological disease. Many lines of preclinical studies demonstrate that neural progenitors committed to dopaminergic fate survive and integrate better than postmitotic DA neurons. We show that the folate-receptor 1 (FolR1),a GPI-anchored cell surface molecule,specifically marks mesDA neural progenitors and immature mesDA neurons. FolR1 expression superimposes with Lmx1a,a bona-fide mesDA lineage marker,during the active phase of mesDA neurogenesis from E9.5 to E14.5 during mouse development,as well as in ESC-derived mesDA lineage. FolR1(+) neural progenitors can be isolated by FACS or magnetic sorting (MAC) which give rise to dopamine neurons expressing TH and Pitx3,whilst FolR1 negative cells generate non-dopaminergic neurons and glia cells. This study identifies FolR1 as a new cell surface marker selectively expressed in mesDA progenitors in vivo and in vitro and that can be used to enrich in vitro differentiated TH neurons. View Publication

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis,and gain-of-function mutations of the receptor are frequently seen in several malignancies,including acute myeloid leukemia,gastrointestinal stromal tumors,and testicular carcinoma. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V),leading to constitutive activation of the receptor. In this study,we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as of activation of downstream signaling pathways including receptor ubiquitination and the Ras/Mek/Erk pathway. Our data demonstrate that,unlike wild-type c-Kit,the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition,we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. We further present evidence that,in contrast to wild-type c-Kit,Src family kinases are dispensable for c-Kit/D816V cell survival,proliferation,and colony formation. Taken together,we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival,thereby contributing to the transforming potential of c-Kit/D816V. View Publication

Introduction: Pathogenic variants in canonical transforming growth factor β (TGFβ) signaling genes predispose patients to thoracic aortic aneurysm and dissection (TAAD),predominantly in aortic root. Although TAAD pathogenesis associated with TGFβ receptor defects is well characterized,distinct and redundant mechanisms of TGFβ isoforms in TAAD incidence and severity remain elusive. Objective: Here we examined the biological role of TGFB2 in smooth muscle cell (SMC) differentiation and investigated how TGFB2 defects can lead to regional TAAD manifestations. Methods: To characterize the role of TGFB2 in SMC differentiation and function,we employed human-induced pluripotent stem cell (hiPSC)-derived SMC differentiation,CRISPR/Cas9 gene editing,three-dimensional SMC constructs,and human aortic tissue samples. Results: Despite the similar effects of different TGFβ isoforms on hiPSC-derived SMC differentiation,siRNA experiments revealed that TGFB2 distinctively displays TGFBR3 dependence for signal transduction,an understudied TGFβ receptor in TAAD. Molecular evaluation of different thoracic aorta regions suggested TGFB2 and TGFBR3 enrichment in the aortic root tunica media. TGFB2 haploinsufficiency (TGFB2KO/+) and TGFB2 neutralization impaired the differentiation of second heart field-derived SMCs. TGFBR3KO/KO prevented the molecular rescue of TGFB2KO/+ by TGFB2 supplementation indicating the involvement of TGFBR3 in TGFB2-mediated SMC differentiation. Lastly,a missense TGFB2 variant (TGFB2G276R/+) caused mechanical defects in SMC tissue ring constructs that were rescued by TGFB2 supplementation or genetic correction. Conclusion: Our data suggests the distinct regulation and action of TGFB2 in SMCs populating the aortic root,while redundant activities of TGFβ isoforms provide implications about the milder TAAD aggressiveness of pathogenic TGFB2 variants. View Publication

Transplantation of human-induced pluripotent stem cell (hiPSC)-derived islet organoids is a promising cell replacement therapy for type 1 diabetes (T1D). It is important to improve the efficacy of islet organoids transplantation by identifying new transplantation sites with high vascularization and sufficient accommodation to support graft survival with a high capacity for oxygen delivery. A human-induced pluripotent stem cell line (hiPSCs-L1) was generated constitutively expressing luciferase. Luciferase-expressing hiPSCs were differentiated into islet organoids. The islet organoids were transplanted into the scapular brown adipose tissue (BAT) of nonobese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice as the BAT group and under the left kidney capsule (KC) of NOD/SCID mice as a control group,respectively. Bioluminescence imaging (BLI) of the organoid grafts was performed on days 1,7,14,28,35,42,49,56,and 63 posttransplantation. BLI signals were detected in all recipients,including both the BAT and control groups. The BLI signal gradually decreased in both BAT and KC groups. However,the graft BLI signal intensity under the left KC decreased substantially faster than that of the BAT. Furthermore,our data show that islet organoids transplanted into streptozotocin-induced diabetic mice restored normoglycemia. Positron emission tomography/MRI verified that the islet organoids were transplanted at the intended location in these diabetic mice. Immunofluorescence staining revealed the presence of functional organoid grafts,as confirmed by insulin and glucagon staining. Our results demonstrate that BAT is a potentially desirable site for islet organoid transplantation for T1D therapy. View Publication

Adenomyosis is also called internal endometriosis and affects about 20{\%} of reproductive-aged women. It seriously reduces life quality of patients because current drug therapies face with numerous challenges. Long-term clinical application of mifepristone exhibits wonderful therapeutic effects with mild side-effects in many disorders since 1982. Since adenomyosis is a refractory disease,we investigate whether mifepristone can be applied in the treatment of adenomyosis. In this study,we investigated the direct effects of mifepristone on human primary eutopic endometrial epithelial cells and stromal cells in adenomyosis. We found that mifepristone causes cell cycle arrest through inhibiting CDK1 and CDK2 expressions and induces cell apoptosis via the mitochondria-dependent signalling pathway in endometrial epithelial cells and stromal cells of adenomyosis. Furthermore,mifepristone inhibits the migration of endometrial epithelial cells and stromal cells through decreasing CXCR4 expression and restricts the invasion of endometrial epithelial cells via suppression of epithelial-mesenchymal transition in adenomyosis. We also found that mifepristone treatment decreases the uterine volume,CA125 concentration and increases the haemoglobin concentration in serum for adenomyosis patients. Therefore,we demonstrate that mifepristone could serve as a novel therapeutic drug in the treatment of adenomyosis,and therefore,the old dog can do a new trick. View Publication

We evaluate the effect of most FDA-approved drugs (>7,000 conditions) on double-strand DNA break repair pathways by analyzing mutational outcomes in human induced pluripotent stem cells. We identify drugs that can be repurposed as inhibitors and enhancers of repair outcomes attributed to non-homologous and microhomology-mediated end joining (NHEJ,MMEJ),and homology-directed repair (HDR). We also identify functions of the proteins estrogen receptor 2 (ESR2) and aldehyde oxidase 1 (AOX1),affecting several key DNA repair proteins,such as ATM and 53BP1. Silencing of ESR2 can have a synergistic effect on increasing HDR when combined with NHEJ inhibition (mean 4.6-fold increase). We further identify drugs that induce synthetic lethality when NHEJ or HDR is blocked and may therefore be candidates for precision medicine. We anticipate that the ability to modulate the DNA repair outcomes with clinically safe drugs will help disease modeling,gene therapy,chimeric antigen receptor immunotherapy,and cancer treatment. DNA repair pathways shape CRISPR editing outcomes. Here,authors identified FDA approved drugs that can be repurposed as repair modulators or to induce synthetic lethality,and uncovered new roles for ESR2 and AOX1 in DNA repair,enhancing editing and offering potential therapeutic applications. View Publication

Genome-wide association studies (GWASs) have increased our knowledge of loci associated with a range of human diseases. However,applying such findings to elucidate pathophysiology and promote drug discovery remains challenging. Here,we created isogenic human ESCs (hESCs) with mutations in GWAS-identified susceptibility genes for type 2 diabetes. In pancreatic beta-like cells differentiated from these lines,we found that mutations in CDKAL1,KCNQ1,and KCNJ11 led to impaired glucose secretion in vitro and in vivo,coinciding with defective glucose homeostasis. CDKAL1 mutant insulin+ cells were also hypersensitive to glucolipotoxicity. A high-content chemical screen identified a candidate drug that rescued CDKAL1-specific defects in vitro and in vivo by inhibiting the FOS/JUN pathway. Our approach of a proof-of-principle platform,which uses isogenic hESCs for functional evaluation of GWAS-identified loci and identification of a drug candidate that rescues gene-specific defects,paves the way for precision therapy of metabolic diseases. View Publication