Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

Abstract

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.

GATA2 mitotic bookmarking is required for definitive haematopoiesis

“…4b ). We observed that expression of the surface marker CD9, an early marker of hemogenic reprogramming 25 , 26 which is also bookmarked by GATA2 (Supplementary Fig. 4e ), was delayed when GATA2 was degraded at M-G1 transition (Fig.…”

“…To gain initial insight regarding the role of GATA2 at M-G1 transition for HSPC generation, we induced hemogenic reprogramming in HDFs with MD-GATA2 or MD mut -GATA2, in combination with GFI1B and FOS 25 , 26 (Fig. 4b ).…”

“…We have previously demonstrated direct reprogramming of mouse 23 , 24 and human fibroblasts 25 , 26 to HSPCs by overexpressing the TFs GATA2, GFI1B and FOS. These TFs induced a dynamic hemogenic process, recapitulating EHT and HSC ontogeny 24 .…”

Application and prospects of somatic cell reprogramming technology for spinal cord injury treatment

“…Thus, fibroblasts have an important advantage for reprogramming because they are easy to obtain and demonstrate multidifferentiation potential. Researchers have successfully transformed fibroblasts into cardiomyocytes ( Bektik and Fu, 2021 ), hematopoietic progenitor cells ( Silverio-Alves et al, 2019 ), neurons, motor neurons ( Son et al, 2011 ; Qin et al, 2018 ; Hu et al, 2019 ), dopaminergic neurons ( Li et al, 2019 ), glutamatergic neurons, GABAergic neurons ( Vierbuchen et al, 2010 ) and oligodendrocyte progenitor cells ( Lee et al, 2018 ). However, fibroblasts have been found to be involved in fibrotic scar formation following acute CNS injury ( Derk et al, 2021 ), indicating their importance in injured spinal cord repair.…”

Cellular reprogramming: Mathematics meets medicine

“…Clinically‐relevant reprogramming successes in laboratory settings have presented exciting potential as well. For example, critical steps have been made toward autologous hematopoetic stem cell therapies for postchemotherapy leukemia patients, as murine committed lymphoid and myeloid progenitors (Riddell et al, 2014) as well as human fibroblasts (Silverio‐Alves, Gomes, Kurochkin, Moore, & Pereira, 2019) were converted into hemogenic cells. This success extends to the field of nerve regeneration therapy, as fibroblasts were reprogrammed into glutaminergic neurons (Yang et al, 2019).…”