Generation Of Megakaryocytes By Chemical Compounds-Induced Lineage Reprogramming And The Underlying Molecular Mechanisms

Platelets are the active product of cytoplasmic fragmentation of mature megakaryocytes in the blood system and play an important role in physiological and pathological processes such as hemostasis,thrombosis,and immune regulation.Platelet transfusion is the most effective strategy for the treatment of thrombocytopenia,but there is currently a serious shortage of platelet sources,mainly due to the reliance on volunteer donations with limited sources,difficulties in preservation,and vulnerability to contamination,which has led to an increasingly acute conflict between platelet supply and demand,and therefore an urgent need to find new sources of platelets.Megakaryocytes,the precursor cells for platelet production,play an important role in hemostasis,coagulation and thrombosis,although their numbers are very small in vivo.In recent years,researchers have worked to induce platelet production from megakaryocytes in vitro to alleviate the great clinical need for platelets,and clinical trial studies have shown that infusion of megakaryocytes obtained in vitro in culture is promising for the treatment of thrombocytopenia.Therefore,in vitro preparation of megakaryocytes has important clinical applications.And this reaserch aimed to find novel seed cell sources to produce megakaryocytes and platelets safely and efficiently.Direct cell lineage reprogramming techniques is able to achieve conversion between cell types through reprogramming strategies such as transcription factors,mi RNAs and compounds.Currently,the technology of cell reprogramming is able to convert somatic cells into pluripotent stem cells and various mature functional cells,which provides a new idea for the acquisition of megakaryocytes.Small molecule compounds not only avoid the risk of gene integration,but also have the advantages of being stable,controllable,inexpensive and safe,providing a safer and more stable way to induce cell reprogramming.A combination of small molecule compounds(4M:Bix01294,RG108,PD0325901,VPA)successfully converted human cord blood erythroblasts(EBs)into induced MKs(i MKs).The reprogramming effect of 4M was validated in this project using erythroblasts as the starting cells.The optimal phase and duration of action of small molecule compounds were determined,the reprogramming system was optimized.Finally a two-stage stepwise induction scheme for the combination of small molecule compounds to induce reprogramming from erythroblasts to megakaryocytes was determined:the first stage was the reprogramming stage,using small molecule compounds and cytokines to induce 7 days to achieve reprogramming from erythroblasts to megakaryocytes;the second stage was the megakaryocyte specialization stage,using cytokine induction to obtain further differentiation and maturation of megakaryocytes.After two stages of induction,the percentage of CD41a~+cells was 84.40%±4.07%on day 14,and the percentage of CD41a~+CD42b~+cells increased significantly,up to 44.60%±4.34%.The induced megakaryocytes(i MKs)obtained by the two-stage reprogramming system have similar cell morphology and ultrastructural characteristics to natural megakaryocytes,express megakaryocyte-specific genes and proteins,and have the ability to secrete megakaryocyte-associated cytokines.The i MKs can form proplatelets with branching structures and release platelets under in vitro platelet-producing conditions.The i MKs-derived platelets have similar ultrastructural features and characteristic protein expression as natural platelets.In vitro functional assays have shown that i MKs-derived platelets can be activated by thrombin and perform adhesion spreading and agglutination functions,functionally resembling natural MKs.To examine the in vivo ability of i MKs to produce platelets,we constructed a thrombocytopenia model in carboplatin-induced severe immunodeficient mice.Based on this model,we found that i MKs migrated to the bone marrow and spleen with blood circulation after infusion into mice,and human-derived platelets could be detected in peripheral blood of mice,and confirmed that platelets released by i MKs in vivo have the ability to participate in thrombosis,indicating that i MKs can differentiate and mature and produce functional platelets in vivo.This study explored the reprogramming mechanisms at the transcriptional level and epigenetic level,respectively.Single-cell transcriptome sequencing was used to classify the cell populations involved in the reprogramming of erythroblasts to megakaryocytes:erythroblasts(EB),erythroid-like cells(EB-like),induced megakaryocytes(i MK-1 and i MK-2),and induced precursors for erythrocytes and megakaryocyte(i PEM).Reprogramming trajectory analysis revealed an efficient cell fate conversion of EBs into i MKs by 4M via the intermediate state of bipotent precursors.Based on the surface markers CD41a and CD235a of precursors for erythrocytes and megakaryocytes reported in the literature,and combined with the highly expressed surface markers CD117 and CD44 of i PEM obtained from single cell transcriptome sequencing analysis,we sorted and purified i PEM with CD235a~+CD41a~+CD117~+CD44~+as sorting markers and induced them to erythrocyte and megakaryocyte,respectively.This study confirmed that i PEM had a bidirectional differentiation potential towards the erythroid lineage and megakaryocytes,and further confirmed that precursors for erythrocytes and megakaryocytes were generated during the reprogramming of erythroblasts to i MKs.ATAC-seq results indicated that extensive chromatin remodeling occurred during reprogramming,with a large number of erythroid-associated motifs turned off early in reprogramming and megakaryocyte-associated motifs in an open state during reprogramming.We verified by q PCR experiments that the expression of megakaryocyte-associated transcription factors GATA2,FLI1,RUNX1,TAL1,GATA1,FOG1,and MEIS1 at the transcriptional level increased with the induction time,and the expression of erythroid-associated transcription factors MYB and KLF1 decreased accordingly.Thus,4M may affect the expression of related transcription factors during reprogramming by participating in the regulation of chromatin accessibility,which in turn drives the cell fate transitionTo further demonstrate the possibility that our combination of small molecule compounds can convert the fate of different cells through cellular reprogramming techniques and consequently obtain novel megakaryocytes more widely,we extended the technical system of reprogramming erythroblasts to megakaryocytes to T cells abundant in blood and found that the system was able to induce the conversion of cord blood and peripheral blood T cells to megakaryocytes,and the proportion of cord blood T cells induced to obtain CD41a~+cells on day 14 was 32.47%±3.37%and The percentage of CD41a~+CD42b~+cells was 13%±0.21%,and the percentage of CD41a~+cells induced by peripheral blood T cells was 4.69%±1.38%,but the percentage of CD41a~+CD42b~+cells was extremely low.We screened AZD4205,a selective inhibitor of JAK1,from a library of signaling pathway-regulating small molecules,based on a high-content cell imaging technology and flow cytometry,to significantly improve the efficiency of T cell to megakaryocyte reprogramming through cascade screening and repeated validation.By optimizing the phase and duration of action of AZD4205,we determined that the optimal protocol for T cell to megakaryocyte reprogramming was:7days of induction using Bix01294,RG108,PD0325901,VPA and AZD4205 combined with cytokines in the first phase,and 7 days of induction using cytokines in the second phase with the removal of small molecule compounds.It was also confirmed that T cell-derived megakaryocytes express megakaryocyte characteristic proteins and are capable of generating polyploid cells,forming proplatelets,and having the ability to release platelets.In summary,the present work establishes and optimizes the reprogramming system of small molecule compounds to induce the reprogramming of erythroblasts to megakaryocyte lineage for the first time,and confirms that the reprogrammed megakaryocytes could produce proplatelets and release functional platelets,functionally resembling natural MKs.The mechanism study revealed that the reprogramming of erythroblasts to i MKs passed through an intermediate state of precursors for erythrocytes and megakaryocytes,and revealed that small molecule compounds may be involved in the regulation of chromatin accessibility by regulating the level of histone modifications during reprogramming and influencing the expression of related transcription factors,which in turn drive the cell fate transition.The induction system of erythroblast to megakaryocyte reprogramming was extended to T cells,which are abundant in source and widely used in clinical practice,and the induction protocol of T cell reprogramming to obtain megakaryocytes was established and optimized to further improve the efficiency and yield of megakaryocyte and platelet preparation in vitro.The chemical small molecule induced lineage reprogramming provides a simple and effective method for obtaining megakaryocytes,and this project improves a novel source of seed cells for obtaining megakaryocytes and platelets safely and efficiently,which is expected to provide a new idea to solve the problem of insufficient supply of clinical platelets,and has important scientific significance and application value..