Chemical Cocktail Induces Hematopoietic Reprogramming And Expands Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells are the most widely used tissue-specific stem cells in clinical practice.Hematopoietic stem cell transplantation remains the one of the most effective treatment of many malignant hematologic disorders.However,the application of hematopoietic stem cells transplantation suffers greatly from the scarcity of HLA-matched donors,which has become a major obstacle regarding the clinical practice of hematopoietic stem cell transplantation.Expansion and reprogramming of hematopoietic stem cells offers two different routs of providing additional transplantable units.Among them,Direct reprogramming from somatic cells to hematopoietic stem cells enables the generation of patient specific HSCs,which in theory provides an inexhaustible source of HSCs for autologous transplantation with minimal chance of autoimmune rejection.The past decade has witnessed great process made in expansion and reprogramming of hematopoietic stem cells mediated by overexpression of transcription factors.However,the conventional method of reprogramming and expansion of hematopoietic stem cells relies on the overexpression of transcription factors,which is mediated by viral vectors and insertion of exogenous genes,causing safety concern for further clinical application.In this study,we used chemical cocktail as a means to expand hematopoietic stem cell and to reprogram differentiated blood cells to hematopoietic stem and progenitor cell like state.Firstly,we isolated GFP~-bone marrow and spleen cells from SCL-t TA/Teto-H2B-GFP double-transgenic mice by Fluorescence-activated cell sorting,and treated these cells with two sets of chemical cocktails which has previously been found in our group to possess the ability of activating the expression of Scl in mouse fibroblasts.Both chemical cocktails activated the expression of Scl in GFP~-blood cells and GFP~-cells were thus reprogrammed into GFP~+cells.We used flow cytometry to further investigate the expression of hematopoietic stem and progenitor cell surface markers in GFP~+cells,and LSK cells were found in GFP~+cells.Colony formation assay showed the GFP~+cells induced by CC1 chemical cocktail exhibitrobust colony forming ability in vitro whereas RNA sequencing revealed the resemblance between the transcriptome of GFP~+cells induced by CC1 and that of the primary hematopoietic stem and progenitor cells.We further tested the reprogramming potential of various hematopoietic cell lineages from the bone marrow.We detected the surface markers of hematopoietic stem and progenitor cells after treatment of CC1 in vitro.It was found that GR1~+cells and CD11b~+cells of the myeloid lineage can be reprogrammed into LSK cells after induction.These LSK cells contain a group of cells with long-term hematopoietic stem cell phenotype.CD3~+T cells of the lymphoid lineage can be reprogrammed into LSK cells,while CD19~+B cells failed to produce LSK cells after CC1 treatment.Colony forming assay showed that CD11b~+macrophage and CD3~+T cell derived LSK cells had clonal multilineage potential.We further speculated whether the the same chemical cocktail has the ability to expand hematopoietic stem cells.We isolated bone marrow LSK cells and treated them with CC1for 7 days in vitro.Compared with the control group,CC1 treatment significantly increased the percentage and absolute number of LSK cells.More importantly,LSK cells expanded by CC1 for seven days still possess multilineage colony forming potential.Both Non-competitive transplantation and competitive transplantation experiment of CC1expanded LSK cells showed significantly higher engraftment rate than those of the control group,even better than that of freshly isolated LSK cells.At last,we treated bone marrow nucleated cells with CC1 for seven days.Cells were collected for single-cell RNA sequencing on day 0,day 1,day 3,day 5 and day 7 respectively,and single-cell RNA sequencing data were analyzed to determine the reprogramming efficiency of diverse lineage cell lines by means of lineage tracing.These data confirmed CC1 chemical cocktail has a double effect on hematopoietic cell reprogramming and HSPC expansion.Therefore,the results of this study provide a potentially different routs to generate hematopoietic stem and progenitor cells.