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Final Report Summary - INDUCING HEMOGENESIS (Mechanisms underlying hemogenic induction in human fibrobalsts)

Hematopoiesis is the best-studied stem/progenitor cell system. It originates from a rare population of multipotent Hematopoietic Stem Cells (HSCs) that give rise to at least ten distinct lineages of mature blood cells. Transplantation of HSCs is a widely utilized therapy for a range of inherited and acquired disorders. Allogeneic transplantation depends on genetic matching to avoid graft rejection and mitigate graft versus host disease as well as host versus graft disease. Even with well-matched grafts failure rates can range from 20-40%. Autologous transplants may be compromised due to previous myeloablative therapies and the nature of the specific diseases. To date, it has been very difficult to maintain or expand HSCs in vitro. Cultured HSCs proliferate but with a rapid loss of stem cell properties. In addition, over two decades of efforts to generate transplantable HSCs from Embryonic Stem Cells (ESCs) or more recently, induced Pluripotent Stem Cells (iPSCs) have met with very limited success. Therefore, there remains a need for alternative strategies to generate HSCs.
The ability to reprogram human somatic cells into somatic stem cells with a degree of multipotency of HSC had not been demonstrated. We have shown that a combination of four transcription factors (Gata2, Gfi1b, cFos and Etv6) efficiently program mouse fibroblasts into a dynamic hemogenic endothelial-like intermediate precursor cells that upon further culture generate clonogenic HSPCs. This process recapitulates in vivo developmental hematopoiesis. The precursor cells generate hematopoietic cells with cell surface and molecular phenotypes very similar to bona fide HSCs.
For inducing pluripotency the transcription factor (TF) combination is remarkably conserved between mouse and human. We hypothesize that hemogenesis can also be induced with a similar combination of TFs in human fibroblasts. In this project we are extending hematopoietic reprogramming to the human system using hemogenic TFs. We are further investigating the mechanisms underlying hemogenic induction in human cells at the level of chromatin and DNA through TF binding, epigenetic profiling, and genome wide expression profiles. Understanding the “mode of action” of these TFs will take us a step closer to generating fully functional patient-specific HSCs for clinical applications. The “Inducing Hemogenesis” project encompass two Specific Aims:

Aim 1. Generation and Molecular Characterization of Human Hemogenic and HSC-like Cells. We are using inducible-systems to generate and extensively characterize the human programmed cells with Gata2, cFos and Gfi1b at various-time points first using cell surface markers and global gene expression analyses. These studies are been complemented by in vitro assays and in vivo engraftment into irradiated immunodeficient recipients (NOD-scid-Il2RG-/-, NSG). To assess self-renewal we will perform secondary transplants with human cells isolated from the bone marrow of primary mice. Umbilical cord blood CD34+ sub-populations were used as a control for gene expression profiling and functional assays. We will further optimizing the induction of hemogenic cells using different combinations of cytokines, cellular and non-cellular substrates and 3D cultures.

Aim 2. Investigation of the Mechanisms that Mediate the Human Hemogenic Reprogramming Process. The ability to recapitulate a dynamic hemogenic process in vitro offers a tractable system to dissect the underlying molecular mechanisms. The binding sites of the TFs GATA2, GFI1B and cFOS will be determined after induction in human fibroblasts using chromatin immunoprecipitation sequencing analysis (ChIP-Seq). These de novo genomic targets will be compared to the established targets in endothelial and hematopoietic cells. These studies will be complemented by the comprehensive genome-wide characterization of epigenetic modifications at the level of chromatin and DNA at different time points after the induction. Both the TF genome location studies and epigenetic profiling will be compared with genome-wide gene expression profiles. For these comparisons we will take advantage of the Encode and Epigenetic Roadmap projects. These projects has systematically mapped genomic elements that regulate transcription, these include regions of transcription, TF association, chromatin structure and histone modifications. The data will be analyzed using sophisticated computational tools and made freely available to the research community. We will use available antibodies for the 3 TFs or proteins tags and histone modifications: H3K27me3 and H3K9me3 (repressive histone marks), H3K4me3 (active promoter mark), H3K36me3 (active elongation mark) and H3K4me1 and H3K27ac (active enhancer marks) and obtain genome-wide maps 2 days and after induction and in CD49f+ cells at days 15 and 25. Input DNA and IgG controls will be also sequenced to define background levels. For analyses of DNA methylation I will use mC-Seq, which involves bisulfite conversion of genomic DNA followed by whole-genome shotgun sequencing.

In summary, our results indicate that hemogenesis can be induced with a similar combination of TFs in human fibroblasts. We have recapitulated our mouse hemogenic programming studies in human adult dermal and neonatal foreskin fibroblasts. The expression of GATA2, cFOS and GFI1B in human fibroblasts is sufficient to generate CD34 and CD49f-positive colonies. We have shown that these colonies contain cells with human LT-HSC phenotype, gene expression signatures and the ability to engraft immunodeficient mice. Genome wide analysis of GATA2, cFOS and GFI1B factor occupancy have identified GATA2 as the dominant factor with independent targeting capacity. Moreover, GATA2 and GFI1B interact and share a cohort of targets that are critical for the specification of definitive hematopoietic stem cells.

The overall innovative aspect of this project is the production of a self-renewing, multipotent HSC-like population from a readily available human cell source (dermal fibroblast). In the future, directly programmed HSCs could provide an unlimited patient-specific source for cell replacement and genetic correction therapies.

More information about the "Inducing Hemogenesis” project can be found here:

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