Here, we report that the functionality of vascular progenitors (VP) generated from normal and disease-primed conventional human induced pluripotent stem cells (hiPSC) can be significantly improved by reversion to a tankyrase inhibitor-regulated human naïve epiblastlike pluripotent state. Naïve diabetic vascular progenitors (N-DVP) differentiated from patient-specific naïve diabetic hiPSC (N-DhiPSC) possessed higher vascular functionality, maintained greater genomic stability, harbored decreased lineage-primed gene expression, and were more efficient in migrating to and re-vascularizing the deep neural layers of the ischemic retina than isogenic diabetic vascular progenitors (DVP). These findings suggest that reprogramming to a stable naïve human pluripotent stem cell state may effectively erase dysfunctional epigenetic donor cell memory or disease-associated aberrations in patientspecific hiPSC. More broadly, tankyrase inhibitor-regulated naïve hiPSC (N-hiPSC) represent a class of human stem cells with high epigenetic plasticity, improved multi-lineage functionality, and potentially high impact for regenerative medicine.

Human pluripotent stem cells (hPSCs) can generate specialized cell lineages that have great potential for regenerative therapies and disease modeling. However, the developmental stage of the lineages generated from conventional hPSC cultures in vitro are embryonic in phenotype, and may not possess the cellular maturity necessary for corrective regenerative function in vivo in adult recipients. Here, we present the scientific evidence for how adult human tissues could generate human–animal interspecific chimeras to solve this problem. First, we review the phenotypes of the embryonic lineages differentiated from conventional hPSC in vitro and through organoid technologies and compare their functional relevance to the tissues generated during normal human in utero fetal and adult development. We hypothesize that the developmental incongruence of embryo-stage hPSC-differentiated cells transplanted into a recipient adult host niche is an important mechanism ultimately limiting their utility in cell therapies and adult disease modeling. We propose that this developmental obstacle can be overcome with optimized interspecies chimeras that permit the generation of adult-staged, patient-specific whole organs within animal hosts with human-compatible gestational time-frames. We suggest that achieving this goal may ultimately have to await the derivation of alternative, primitive totipotent-like stem cells with improved embryonic chimera capacities. We review the scientific challenges of deriving alternative human stem cell states with expanded embryonic potential, outline a path forward for conducting this emerging research with appropriate ethical and regulatory oversight, and defend the case of why current federal funding restrictions on this important category of biomedical research should be liberalized.

Vascular regenerative therapies with conventional human induced pluripotent stem cells (hiPSC) currently remain limited by high interline variability of differentiation and poor efficiency for generating functionally transplantable vascular progenitors (VP). Here, we report the advantage of tankyrase inhibitor-regulated naïve hiPSC (N-hiPSC) for significantly improving vascular cell therapies. Conventional hiPSC reprogrammed from type-1 diabetic donor fibroblasts (DhiPSC) were stably reverted to naïve epiblast-like state with high functional pluripotency with a cocktail of LIF and three small molecules inhibiting the tankyrase, MEK, and GSK3b signaling pathways (LIF-3i). Naïve diabetic VP (N-DVP) differentiated from naïve DhiPSC (N-DhiPSC) expanded more efficiently, possessed higher proliferation, possessed more stable genomic integrity and displayed higher in vitro vascular functionality than primed diabetic VP (DVP) generated from isogenic conventional DhiPSC.Moreover, N-DVP survived, migrated, and engrafted in vivo into the deep vasculature of the neural retinal layers with significantly higher efficiencies than isogenic primed DVP in a murine model of ischemic retinopathy. Epigenetic analyses of CpG DNA methylation and histone configurations at developmental promoters of N-hiPSC revealed tight regulation of lineage-specific gene expression and a de-repressed naïve epiblast-like epigenetic state that was highly poised for multi-lineage transcriptional activation. We propose that reprogramming of patient donor cells to a tankyrase inhibitor-regulated N-hiPSC may more effectively erase epigenetic aberrations sustained from chronic diseases such as diabetes for subsequent regenerative therapies. More broadly, tankyrase inhibitor-regulated N-hiPSC represent a new class of human stem cells with high epigenetic plasticity, improved multilineage functionality, and potentially high impact for regenerative medicine.

C/EBPα mediates myeloid differentiation, and its reduced activity is central to myeloid transformation (Friedman Int. J. Hematol. 2015). The murine Cebpa gene contains a 450 bp, +37 kb element that acquires the enhancer-specific H3K4me1 and H3K27Ac histone marks as LT-HSC progress to GMP and directs hCD4 transgene expression to GMP and myeloid CFUs (Guo et al. Blood 2012; Guo et al. J. Leuk. Biol. 2014). Moreover, CRISPR/Cas9 mediated, biallelic replacement of the enhancer with a variant harboring mutations in its seven Ets sites reduces Cebpa RNA >10-fold in 32Dcl3 cells (Cooper et al. PLoS One 2015), and germline or Cre-mediated enhancer deletion leads to marked reduction in Cebpa RNA in marrow LSK, CMP, and GMP, with 3-fold reduction in GMP, CFU-G, and neutrophils and indefinite myeloid colony replating in IL-3, a preleukemic phenotype (Guo et al. PLoS One 2016; Avellino et al. Blood 2016). The enhancer contains four conserved RUNX1 cis elements that bind RUNX1 in gel shift or ChIP assays, Runx1 gene deletion reduces Cebpa RNA 5-fold in Lin- and 2-fold in GMP marrow cells, Runx1 cis element mutation reduces luciferase reporter activity 6-fold in 32Dcl3 myeloid cells (Guo et al. Blood 2012), and in AMLs with t(8;21) RUNX1-ETO interacts with the human CEBPA locus at the homologous +42 kb enhancer (Ptasinska et al. Leukemia 2012). Mutation of the CEBPA enhancer has not been seen in human AML cases, perhaps reflecting preference for upstream pathway alteration to suppress both alleles and additional genes. As a further example of a leukemic alteration affecting enhancer activity, elevated EVI1 is a high-risk feature in AML, and EVI1 binds and represses the +37 kb Cebpa enhancer (Wilson et al. J. Biol. Chem. 2016). We have characterized additional pathways whose modulation in AML may reduce CEBPA enhancer activity. Deletion of the Pu.1 -14 kb enhancer leads to AML in mice, and PU.1 binds the Cebpa enhancer in ChIP and gel shift; we now find that mutation of the one Cebpa enhancer Ets site that binds PU.1 reduces reporter activity 4-fold in 32Dcl3 cells, providing the first functional evidence that PU.1 regulates the enhancer. ~10% of human AMLs harbor mutant C/EBPα proteins, CEBPα binds the enhancer in ChIP and gel shift, and mutation of the two enhancer C/EBP elements reduces reporter activity; we have now used CRISPR/Cas9 to generate a 32Dcl3 cell line with the two enhancer C/EBPsites mutated in one allele and find 60% reduction in Cebpa RNA expression. FLT3/ITD is a constitutively activated mutant form of the receptor tyrosine kinase found in ~30% of AML cases and confers high risk. While FLT3/ITD predominantly provides proliferative signals, it may also contribute to impaired differentiation, as FLT3/ITD inhibition in AML cases leads to a neutrophilic differentiation syndrome. We previously demonstrated that FLT3/ITD reduces Cebpa RNA in 32Dcl3 cells and patient leukemic blasts and that the first generation FLT3/ITD inhibitor lestaurtinib increases Cebpa; we now find that the effect on Cebpa RNA is also reversed in 32Dcl3 cells by the next generation inhibitor crenolanib, that FLT3/ITD reduces Cebpa enhancer H3K4me1 and H3K27Ac histone marks several-fold, and that both of these activating marks are also restored by crenolanib. FLT3/ITD signaling leads to C/EBPα serine phosphorylation to reduce its activity, which may account in part for the inhibitory effect of FLT3/ITD on Cebpa enhancer activity. Finally, NUP98-HOX fusion proteins contribute to a small percent of AML cases. Vav-NUP98/HOXD13 (NHD13) mice develop MDS and can progress to AML; we now find that Cebpa RNA is reduced 5-fold in Vav-NHD13 CMP and LSK, with 5-fold increased HoxA9 RNA, that Vav-NHD13::Cebpa Enh-hCD4 compound heterozygous mice have reduced hCD4 expression in GMP and CMP, implicating a direct effect on enhancer activity, and that expression of NHD13 in 32Dcl3 subclones prevents G-CSF induction of Cebpa, MPO, and morphologic differentiation. These 32Dcl3 lines retain levels of Gcsfr equal to empty vector-transduced cells in IL-3 and manifest increased HoxA9 in response to G-CSF. Moreover, HoxA9 binds the Cebpa enhancer in ChIP using marrow-derived HoxA9/Meis1 myeloid lines. Potentially, NHD13 induces HoxA9, which then binds the Cebpa enhancer directly or via other factors to repress transcription. Identifying mechanisms that repress CEBPA hematopoietic enhancer activity in AML may identify approaches to induce differentiation. Disclosures No relevant conflicts of interest to declare.