We have developed and characterized an optimized reprogramming system that generates high quality hiPSC from human myeloid progenitors with unprecedented efficiency. To our knowledge, this study is the first to identify important synergies between hematopoietic regulatory circuits activated by GFs and extrinsic niche factors to efficiently direct the pluripotency induction of lineage-committed myeloid cells. Efficient reprogramming correlated not simply to endogenous expression of individual Core reprogramming factors (e.g., SOX2, OCT4, NANOG), or their downstream Core module targets, but to ESC-like expression levels of epigenetic regulators (e.g., PcG/PRC2 complex, MYC complex, and Trithorax complex) and their downstream targets (e.g., ESC, MYC, PRC1, and PRC2 modules). Collectively, these ESC circuits were poised in partially reprogrammed expression states following GF activation of myeloid progenitors, prior to ectopic episomal Yamanaka factor expression (). The episomal reprogramming efficiency of BMSC-primed progenitors, which was ~4–10% in unfractionated day 0 CB cells, and ~50–65% in purified episome-expressing myeloid cells was 4 to 5 logs greater than that of fibroblasts or keratinocytes. Our GFP purification experiments demonstrated that AP+ hESC-like colonies were emerging not from a minority population, but from the majority of episome-expressing myeloid cells. Moreover, the great majority (~50–80%) of these hESC-like colonies had already achieved a fully reprogrammed NANOG+TRA-1-81+ (Type III hiPSC) phenotype by 3–5 weeks following 4F or 7F episomal nucleofection. The application of methods with higher gene transfer efficiencies for expressing reprogramming factors (e.g. via synthetic mRNAs or microRNAs) may allow further optimization of this hematopoietic reprogramming system. More importantly, this experimental system opens new avenues of molecular, epigenetic and proteomic investigations for elucidating novel micro-environmental factors that drive rapid and efficient reprogramming in synchronized populations of donor cells in more defined conditions.
Efficient episomal reprogramming of human myeloid progenitors is mediated by synergies between extrinsic stromal signals and the GF activation of partially reprogrammed ESC-like gene modules.
Our demonstration that lineage-committed CD33+
myeloid cells, and not immature hematopoietic stem-progenitors were more efficiently reprogrammed contrasts and refines the conclusions of previous studies that suggested primitive stem-progenitors are the most amenable cell type for factor-driven reprogramming 
. In an inducible transgenic ‘secondary system’, Eminli et al 
similarly observed that lineage-committed myeloid progenitors (e.g
., GMP, CMP, and MEP populations) had the highest reprogramming potential of all hematopoietic cell types (including primitive HSC), and that this propensity was not directly related to their proliferative status. In agreement with our findings, several somatic cell nuclear transfer reprogramming studies also reported that undifferentiated HSC did not possess a greater cloning efficiency than differentiated myeloid cells 
. Although, the cellular and molecular nature of facile myeloid reprogramming requires further investigation, it is likely related to the unique epigenetic plasticity of hematopoietic progenitors which consists of a CpG methylome, a highly dynamic chromatin structure (e.g.
transcriptionally-active (H3-K4me3), and repressive (H3-K27me3) histone marks 
) that may be similar in configuration to ESC. Collectively, our results are consistent with the notion that the genome and epigenome of committed myeloid progenitors possess a transcriptionally permissive ESC-like state. The Yamanaka factors appeared to cooperate with soluble and contact-dependent stromal signals to further accelerate conversion of GF-activated ESC-like myeloid circuits to a stable pluripotent state. Accordingly, bone marrow stroma are known to increase the ‘stemness’ of in vitro
cultured hematopoietic progenitors by increasing expression levels of enzymes and pathways involved in self-renewal (e.g., DNMT3A, DNMT3B, TERT,
NOTCH, and WNT), and by promoting epigenetic ESC-like histone modifications 
. Our systematic GSEA of BMSC-primed myeloid cells confirmed that stromal priming differentially activated multiple soluble and cell contact-dependent pathways that have previously been previously implicated in potentiating ESC self-renewal and induced pluripotency including WNT activation, p53 destabilization, and integrin signaling 
. Additionally, BMSC-primed CB cells possessed unexpected, yet statistically significant molecular activation of Toll receptor and NFκB signaling pathways, which have previously been implicated in regulating pluripotency 
. However, the mechanism by which activation of NFκB signaling by BMSC priming mediated augmentation of factor-driven reprogramming in myeloid cells remains to be elucidated. Interestingly, a recent study has independently discovered a role for TLR signaling in driving efficient viral vector-mediated reprogramming 
An important theme unveiled from our studies was that efficient induced pluripotency might require extrinsic micro-environmental activation of a molecular framework that commonly regulates self-renewal and differentiation in both hematopoietic progenitors and ESC. Hematopoietic progenitors and ESCs share common transcriptional programs that allow them to self-renew while remaining poised to differentiate into multiple cell lineages 
. In ESC, self-renewal is regulated by concerted networks that include active MYC-regulated circuits, inactive Polycomb-regulated circuits, and an OCT4-interacting epigenetic network 
. The generation of iPSC by defined factors requires the epigenetic activation of all of these transcriptional networks 
, which are all normally quiescent in somatic fibroblasts. We demonstrated that GF-activated myeloid progenitors already possessed high hESC-like expression levels of all these regulatory circuits (except the Core module) in partially reprogrammed transcriptional states (). These pre-activated networks were rapidly reconfigured from hematopoietic to ESC-like transcriptional patterns following episomal expression of the Yamanaka core factors (e.g.
OCT4, SOX2). Furthermore the reconfiguration of these de novo
activated circuits networks to hESC-like patterns was dramatically accelerated by extrinsic stromal signals with an unprecedented bulk efficiency and rapidity that was limited only by the efficiency of episomal gene transfer.
MYC and its downstream transcriptional networks (ESC and MYC modules) appeared instrumental for efficient myeloid reprogramming, consistent with its role as a master regulator of chromatin modification, and its maintenance of the pluripotent state through effects on histone acetyltransferases 
. Additionally, activation of MYC and its networks is a known downstream target of JAK-STAT3 signaling by the GFs implicated in these studies (e.g
., FLT3, TPO, PDGF, CCL2). Furthermore, many of the 334 genes in the MYC-regulated ESC module are established transcriptional regulators of HSC self-renewal (e.g
., MYC, DNMT1, and HDAC1). However, the promoters of many of these ESC module genes also bind the ESC-specific Core factors (SOX2, OCT4, and NANOG; SON ), thus likely providing a link for cross-regulation of Core and PcG networks 
. Moreover, MYC-regulated circuits were progressively expressed in hematopoietic progenitors of decreasing maturity, thus providing one basis for the known augmented reprogramming efficiency seen in developmentally immature somatic cells.
Unexpectedly, GF-activated myeloid cells also expressed ESC-like levels of an organized OCT4-associated circuit 
that is known to interact directly and support the function of the Core reprogramming factors in ESC. The role and function of this OCT4-interacting regulatory circuit has not yet been described in any class of stem cell or progenitor other than ESC. In a variety of somatic cells including fibroblasts, the activation of the Core network by defined factors is a rate-limiting step, and requires the additional epigenetic activation of a large auxiliary network of DNA and chromatin modifying proteins. These networks physically interact with OCT4 and NANOG in a protein-protein interactome to support pluripotency 
. This OCT4 interactome includes a wide repertoire of ESC factors that have been experimentally validated to facilitate iPSC generation (e.g.,
PcG, SWI/SWF, Trithorax, NURD, Chromodomain, LSD1 complexes), and that interact directly with the Core factors to regulate self-renewal, lineage fate, and promotion of hyper-transcriptional chromatin. The surprising observation that an OCT4-associated network was already active in GF-primed CB myeloid progenitors suggests a common regulatory biology of self-renewal and differentiation in both ESC and hematopoietic progenitors. Consistent with our hypothesis that myeloid progenitors already possess the required molecular infrastructure of induced pluripotency, but lack only activation of core circuits (by ectopic episomal core factor expression), previous studies demonstrated that CB progenitors can be reprogrammed (albeit inefficiently) with only SOX2 and OCT4 transgenes 
In summary, this highly optimized human myeloid reprogramming system provides a new paradigm for understanding the augmented reprogramming capacity of somatic progenitors. Detailed elucidation of the stromal signals that augmented myeloid reprogramming should facilitate highly efficient reprogramming in defined conditions for other cell types, thus obviating the need for stromal priming. Further dissection of this experimental system may also unveil a common biology that regulates hematopoietic progenitors and pluripotent stem cells.