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Transcriptional landscape of direct reprogramming toward hematopoietic stem cells

ABSTRACT: Transcriptional landscape of direct reprogramming toward hematopoietic stem cells

PROVIDER: PRJNA1194990 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR31632206_1.fastq.gz	Fastqsanger.gz
	SRR31632207_1.fastq.gz	Fastqsanger.gz
	SRR31632208_1.fastq.gz	Fastqsanger.gz

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Transcriptional landscape of direct reprogramming toward hematopoietic stem cells

Project description:Direct reprogramming of human fibroblasts into hematopoietic stem cells (HSCs) offers a promising strategy for generating autologous cells to treat blood and immune disorders. However, low conversion efficiency and incomplete characterization of reprogrammed states limit current protocols. To address this, we assembled a single-cell transcriptomic reference atlas spanning hematopoietic, endothelial, and fibroblast cell types, and evaluated a novel algorithmically predicted transcription factor (TF) recipe—GATA2, GFI1B, FOS, REL, and STAT5A—for induction of HSC-like states. The 5TF recipe increased conversion efficiency of fibroblasts to CD34+ cells by approximately three-fold compared to previous protocols. Long-read single-cell RNA sequencing revealed heterogeneous expression of hematopoietic- and endothelial-associated genes, consistent with partial reprogramming. To benchmark re- programmed cells, we developed an approach to map their transcriptomic positions relative to native and initial cell states within the reference atlas. Finally, we cataloged isoform diversity in reprogrammed cells, suggesting a role for alternative splicing in reprogramming dynamics. These findings highlight both the promise and complexity of direct HSC reprogramming and provide a framework for optimizing future protocols.

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Reprogramming of human fibroblasts toward a cardiac fate

Project description:Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or microRNAs has recently been demonstrated. The potential clinical applicability of these findings is based on the minimal regenerative potential of the adult human heart and the limited availability of human heart tissue. An initial, but mandatory step toward clinical application of this approach is to establish conditions for conversion of adult human fibroblasts to a cardiac phenotype. Toward this goal, we sought to determine the optimal combination of factors necessary and sufficient for direct myocardial reprogramming of human fibroblasts. Here we show that four human cardiac transcription factors, including Gata4, Hand2, Tbx5, and myocardin, and two microRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts. After maintenance in culture for 4-11 weeks, human fibroblasts reprogrammed with these proteins and microRNAs displayed sarcomere-like structures and calcium transients, and a small subset of such cells exhibited spontaneous contractility. These phenotypic changes were accompanied by expression of a broad range of cardiac genes and suppression of non-myocyte genes. These findings indicate that human fibroblasts can be reprogrammed to cardiac-like myocytes by forced expression of cardiac transcription factors with muscle-specific microRNAs and represent a step toward possible therapeutic application of this reprogramming approach. Human foreskin fibroblasts were transduced with 5 transcription factors and total RNA was obtained 4 weeks later. total RNA was also obtained from human foreskin fibroblasts as a negative control and adult human heart tissue as a positive control. The expression level of genes in each sample was compared.

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