Hemophilia A ameliorated in mice by CRISPR-based in vivo genome editing of human Factor VIII.

Hemophilia A is a monogenic disease with a blood clotting factor VIII (FVIII) deficiency caused by mutation in the factor VIII (F8) gene. Current and emerging treatments such as FVIII protein injection and gene therapies via AAV-delivered F8 transgene in an episome are costly and nonpermanent. Here, we describe a CRISPR/Cas9-based in vivo genome editing method, combined with non-homologous end joining, enabling permanent chromosomal integration of a modified human B domain deleted-F8 (BDD-F8) at the albumin (Alb) locus in liver cells. To test the approach in mice, C57BL/6 mice received tail vein injections of two vectors, AAV8-SaCas9-gRNA, targeting Alb intron 13, and AAV8-BDD-F8. This resulted in BDD-F8 insertion at the Alb locus and FVIII protein expression in the liver of vector-, but not vehicle-, treated mice. Using this approach in hemophilic mice, BDD-F8 was expressed in liver cells as functional human FVIII, leading to increased plasma levels of FVIII and restoration of blood clotting properties in a dose-dependent manor for at least 7 months, with no detectable liver toxicity or meaningful off-target effects. Based on these findings, our BDD-F8 genome editing approach may offer an efficacious, long-term and safe treatment for patients with hemophilia A.

sRNA-seq analysis of human embryonic stem cells and definitive endoderm reveals differentially expressed microRNAs and novel IsomiRs with distinct targets.

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem-loop for miR-302a, an important miRNA for both hESCs self-renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem-loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR-302 cluster as a model system. A miRNA mimetic for miR-302a-5p, but not miR-302a-5p(+3), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a-5p(+3) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs.

Is stage-specific embryonic antigen 4 a marker for human ductal stem/progenitor cells?

The presence of pancreatic stem cells (PnSCs) has not been firmly demonstrated in the human or animal pancreas. Previous reports have suggested that ductal and acinar structures in the exocrine pancreas can be a potential source of progenitor cells. More recently, immature insulin precursors in the periphery of human islets have been found to self-replicate and differentiate to endocrine cells in vitro. Transplantation of these cells under the kidney capsule improves the diabetic state in mice. The controversy surrounding where PnSCs reside could be resolved if a specific marker were to be found that allowed their identification, purification, and directed differentiation to endocrine cells. We have identified in human pancreas cells positive for the stage-specific embryonic antigen 4 (SSEA4), a stem cell marker. These cells also express ductal, pancreatic progenitor, and stem cell protein markers. Interestingly, some of the SSEA4(+) cells scattered in the ducts do not show a ductal cell phenotype. SSEA4(+)-sorted cells formed aggregate-like spheres in culture and robustly differentiated to pancreatic hormone-expressing cells in conditions of high glucose concentration and B27 supplementation. We hypothesize that SSEA4(+) cells or a subpopulation of those cells residing in the pancreatic ducts may be the elusive PnSCs, and in this case, SSEA4 may represent a potential surface antigen marker for human PnSCs. The discovery of specific markers for the identification and purification of human PnSCs would greatly facilitate studies aimed at the expansion of these cells and the development of targeting tools for their potential induction to insulin-producing cells.

Inhibitors of Src and focal adhesion kinase promote endocrine specification: impact on the derivation of ß-cells from human pluripotent stem cells.

Stepwise approaches for the derivation of ß-cells from human embryonic stem cells have been described. However, low levels of endocrine specification limit the final yield of insulin-producing ß-cells. In this study, we show that the pyrrolo-pyrimidine Src family kinase (SFK) inhibitor PP2 effectively promotes the endocrine specification of human embryonic stem cell derivatives based on its capacity to induce the expression of proendocrine transcription factors (NGN3, NEUROD1, NKX2.2, and PAX4) and to significantly increase the final yield of insulin-positive cells. We further demonstrate that PP2 inhibits the activation of focal adhesion kinase (FAK), and selective inhibition of this kinase is also sufficient to induce early endocrine commitment based on increased expression of NGN3, NEUROD1, and NKX2.2. Additional studies using dominant negative constructs and isolated human fetal pancreata suggest that c-Src is at least partially responsible for inhibiting early endocrine specification. Mechanistically, we propose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGFßR/Smad2/3 pathway. Moreover, we show that inhibition of SFK/FAK signaling suppresses cell growth, increases the expression of the ß-cell-associated cyclin-dependent kinase inhibitor p57kip2, and simultaneously suppresses the expression of Id1 and Id2. This study has important implications for the derivation of ß-cells for the cell-based therapy of diabetes and sheds new light on the signaling events that regulate early endocrine specification.

A distinct microRNA signature for definitive endoderm derived from human embryonic stem cells.

Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs.

A novel approach for the derivation of putative primordial germ cells and sertoli cells from human embryonic stem cells.

Using human embryonic stem cells (hESCs), we describe a novel method for the rapid derivation and enrichment of cells that are comparable to primordial germ cells (PGCs) and Sertoli cells. The methodology described is based on modest changes to the growth conditions commonly used to expand hESCs and does not require genetic manipulation or complex three-dimensional culture. Remarkably, we have determined that simply reducing the size of cultured ESC colonies and manipulating the number of feeding cycles, results in the rapid emergence of cells that are comparable to migratory PGCs. Importantly, these cells can be monitored and purified on the basis of the expression of the chemokine receptor CXCR4. Under more stringent differentiating conditions these cells mature and upregulate the expression of specific germ cell markers. Importantly, this process is accompanied by the development of Sertoli-like support cells. Such cells normally provide trophic support and immunoprotection to developing germ cells and may have significant clinical utility in the prevention of graft rejection. The putative Sertoli-germ cell cocultures generated in this study may ultimately be developed to study and manipulate interactions and processes involved in human gametogenesis.