Spliceosome inhibitor induces human hematopoietic progenitor cell reprogramming toward stemness.

The application of hematopoietic stem cells (HSCs) has been restricted due to limited cell sources and conventional methods for generating these cells by cell expansion and pluripotent stem cell differentiation have not been clinically achieved. Cell reprogramming technique provides a new hope for generating desirable cells. We previously reported that mouse differentiated hematopoietic cell reprogramming could be induced by small molecule compounds to generate hematopoietic stem/progenitor-like cells, whether the human hematopoietic cells could also be reprogrammed into HSCs by chemical compounds remains elusive. Here, we demonstrated for the first time that human committed hematopoietic progenitors could be reprogrammed into multipotent progenitors by spliceosome inhibitor. Combination of single cell RNA-sequencing and genetic lineage tracing including exogenous barcodes and endogenous mitochondrial DNA mutations confirmed the reprogramming procession. Although the small chemical compound inhibiting spliceosome function only induces the differentiated hematopoietic progenitors to acquire plasticity and reprograms them into multipotent progenitors but not stem cells so far, this study still provides a proof-of-concept strategy for generating HSCs based on combining two independent steps together in future, first differentiating rare HSCs into large number of progenitors then reprogramming these progenitors into huge number of HSCs. Further dissecting the mechanism underlying spliceosome inhibitor-induced human hematopoietic cell reprogramming in future will help us comprehensively understanding not only the chemical reprogramming to generate desirable human cells for clinical translation but also hematopoiesis under physiological and pathological conditions.

Trajectory of chemical cocktail-induced neutrophil reprogramming.

Hematopoietic reprogramming holds great promise for generating functional target cells and provides new angle for understanding hematopoiesis. We reported before for the first time that diverse differentiated hematopoietic cell lineages could be reprogrammed back into hematopoietic stem/progenitor cell-like cells by chemical cocktail. However, the exact cell types of induced cells and reprogramming trajectory remain elusive. Here, based on genetic tracing method CellTagging and single-cell RNA sequencing, it is found that neutrophils could be reprogrammed into multipotent progenitors, which acquire multi-differentiation potential both in vitro and in vivo, including into lymphoid cells. Construction of trajectory map of the reprogramming procession shows that mature neutrophils follow their canonical developmental route reversely into immature ones, premature ones, granulocyte/monocyte progenitors, common myeloid progenitors, and then the terminal cells, which is stage by stage or skips intermediate stages. Collectively, this study provides a precise dissection of hematopoietic reprogramming procession and sheds light on chemical cocktail-induction of hematopoietic stem cells.

Hematopoietic Reprogramming Entangles with Hematopoiesis.

Hematopoiesis generally refers to hematopoietic development in fetuses and adults, as well as to hematopoietic stem cell differentiation into progeny lineages. The multiple processes that generate diverse hematopoietic cells have been considered to be unidirectional. However, many reports have recently demonstrated that these processes are not only reversible but also interconvertible via cell reprogramming. The cell reprogramming that occurs in hematopoietic cells is termed hematopoietic reprogramming. We focus on both autogenous and artificial hematopoietic reprogramming under physiological and pathological conditions that is mainly directed by the actions of transcription factors (TFs), chemical compounds, or extracellular cytokines. A comprehensive understanding of hematopoietic reprogramming will help us not only to generate desirable cells for cell therapy but also to further analyze normal and malignant hematopoiesis.

Chemical Cocktail Induces Hematopoietic Reprogramming and Expands Hematopoietic Stem/Progenitor Cells.

Generation of hematopoietic stem/progenitor cells (HSPCs) via cell expansion or cell reprogramming has been widely achieved by overexpression of transcription factors. Herein, it is reported that without introducing exogenous genes, mouse fibroblasts can be reprogrammed into hemogenic cells based on lineage tracing analysis, which further develop into hematopoietic cells, by treatment of cocktails of chemical compounds. The chemical cocktails also reprogram differentiated hematopoietic cells back into HSPC-like cells. Most importantly, the chemical cocktails enabling hematopoietic reprogramming robustly promote HSPC proliferation ex vivo. The expanded HSPCs acquire enhanced capacity of hematopoietic reconstruction in vivo. Single-cell sequencing analysis verifies the expansion of HSPCs and the cell reprogramming toward potential generation of HSPCs at the same time by the chemical cocktail treatment. Thus, the proof-of-concept findings not only demonstrate that hematopoietic reprogramming can be achieved by chemical compounds but also provide a promising strategy for acquisition of HSPCs by chemical cocktail-enabled double effects.

Reprogramming of Fibroblasts to Neural Stem Cells by a Chemical Cocktail.

Chemically induced cell fate conversion, including reprogramming to pluripotent stem cells and direct reprogramming to somatic cells, has been proved to be an alternative strategy with many advantages, in comparison with conventional transcription factors- or microRNAs-enabled cell reprogramming. Many functional and desirable cells have been generated via the chemically induced reprogramming. Neural stem cells (NSCs) hold great potential in basic research and clinical application. Here, we describe a detailed protocol for converting mouse fibroblasts into NSCs by a cocktail of chemical compounds.