RESUMO
The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.
Assuntos
Reprogramação Celular , Fibroblastos/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Adesão Celular , Diferenciação Celular , Humanos , Camundongos , Fatores de Transcrição/genéticaRESUMO
Transcription factor (TF)-based reprogramming of somatic tissues holds great promise for regenerative medicine. Previously, we demonstrated that the TFs GATA2, GFI1B, and FOS convert mouse and human fibroblasts to hemogenic endothelial-like precursors that generate hematopoietic stem progenitor (HSPC)-like cells over time. This conversion is lacking in robustness both in yield and biological function. Herein, we show that inclusion of GFI1 to the reprogramming cocktail significantly expands the HSPC-like population. AFT024 coculture imparts functional potential to these cells and allows quantification of stem cell frequency. Altogether, we demonstrate an improved human hemogenic induction protocol that could provide a valuable human in vitro model of hematopoiesis for disease modeling and a platform for cell-based therapeutics. DATABASE: Gene expression data are available in the Gene Expression Omnibus (GEO) database under the accession number GSE130361.
Assuntos
Diferenciação Celular/genética , Reprogramação Celular/genética , Hemangioblastos/citologia , Células-Tronco Hematopoéticas/citologia , Animais , Técnicas de Cocultura/métodos , Fibroblastos/citologia , Fibroblastos/metabolismo , Fator de Transcrição GATA2/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Hemangioblastos/metabolismo , Hematopoese/genética , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Repressoras/genética , Fatores de Transcrição/genéticaRESUMO
During development, hematopoietic stem and progenitor cells (HSPCs) arise from specialized endothelial cells by a process termed endothelial-to-hematopoietic transition (EHT). The genetic program driving human HSPC emergence remains largely unknown. We previously reported that the generation of hemogenic precursor cells from mouse fibroblasts recapitulates developmental hematopoiesis. Here, we demonstrate that human fibroblasts can be reprogrammed into hemogenic cells by the same transcription factors. Induced cells display dynamic EHT transcriptional programs, generate hematopoietic progeny, possess HSPC cell surface phenotype, and repopulate immunodeficient mice for 3 months. Mechanistically, GATA2 and GFI1B interact and co-occupy a cohort of targets. This cooperative binding is reflected by engagement of open enhancers and promoters, initiating silencing of fibroblast genes and activating the hemogenic program. However, GATA2 displays dominant and independent targeting activity during the early phases of reprogramming. These findings shed light on the processes controlling human HSC specification and support generation of reprogrammed HSCs for clinical applications.
Assuntos
Reprogramação Celular , Hemangioblastos/citologia , Hemangioblastos/metabolismo , Fatores de Transcrição/metabolismo , Adulto , Sequência de Bases , Elementos Facilitadores Genéticos/genética , Fibroblastos/metabolismo , Fator de Transcrição GATA2/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Recém-Nascido , Fenótipo , Regiões Promotoras Genéticas/genética , Ligação ProteicaRESUMO
Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li-Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a ß-catenin-independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.