RESUMEN
The classical model of hematopoietic hierarchies is being reconsidered on the basis of data from in vitro assays and single cell expression profiling. Recent experiments suggested that the erythroid lineage might differentiate directly from multipotent hematopoietic stem cells / progenitors or from a highly biased subpopulation of stem cells, rather than transiting through common myeloid progenitors or megakaryocyte-erythrocyte progenitors. We genetically barcoded autologous rhesus macaque stem and progenitor cells, allowing quantitative tracking of the in vivo clonal output of thousands of individual cells over time following transplantation. CD34+ cells were lentiviral-transduced with a high diversity barcode library, with the barcode in an expressed region of the provirus, allowing barcode retrieval from DNA or RNA, with each barcode representing an individual stem or progenitor cell clone. Barcode profiles from bone marrow CD45-CD71+ maturing nucleated red blood cells were compared with other lineages purified from the same bone marrow sample. There was very high correlation of barcode contributions between marrow nucleated red blood cells and other lineages, with the highest correlation between nucleated red blood cells and myeloid lineages, whether at earlier or later time points post transplantation, without obvious clonal contributions from highly erythroid-biased or restricted clones. A similar profile occurred even under stressors such as aging or erythropoietin stimulation. RNA barcode analysis on circulating mature red blood cells followed over long time periods demonstrated stable erythroid clonal contributions. Overall, in this nonhuman primate model with great relevance to human hematopoiesis, we documented continuous production of erythroid cells from multipotent, non-biased hematopoietic stem cell clones at steady-state or under stress.
Asunto(s)
Eritropoyesis , Células Madre Hematopoyéticas , Animales , Diferenciación Celular , Células Cultivadas , Hematopoyesis , Macaca mulatta , Células Madre MultipotentesRESUMEN
Lentiviral vectors (LVs) are used for delivery of genes into hematopoietic stem and progenitor cells (HSPCs) in clinical trials worldwide. LVs, in contrast to retroviral vectors, are not associated with insertion site-associated malignant clonal expansions and, thus, are considered safer. Here, however, we present a case of markedly abnormal dysplastic clonal hematopoiesis affecting the erythroid, myeloid, and megakaryocytic lineages in a rhesus macaque transplanted with HSPCs that were transduced with a LV containing a strong retroviral murine stem cell virus (MSCV) constitutive promoter-enhancer in the LTR. Nine insertions were mapped in the abnormal clone, resulting in overexpression and aberrant splicing of several genes of interest, including the cytokine stem cell factor and the transcription factor PLAG1. This case represents the first clear link between lentiviral insertion-induced clonal expansion and a clinically abnormal transformed phenotype following transduction of normal primate or human HSPCs, which is concerning, and suggests that strong constitutive promoters should not be included in LVs.
Asunto(s)
Terapia Genética/métodos , Vectores Genéticos/uso terapéutico , Hematopoyesis/genética , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/virología , Lentivirus/genética , Transducción Genética , Animales , Antígenos CD34/metabolismo , Células Clonales , Terapia Genética/efectos adversos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Sustancias Luminiscentes/metabolismo , Macaca mulatta , Mutagénesis Insercional/genética , Regiones Promotoras Genéticas , Empalme de Proteína/genética , Secuencias Repetidas Terminales/genética , Trasplante AutólogoRESUMEN
Active regulatory elements in hematopoietic stem cells (HSCs) are incompletely characterized, since extant approaches immunophenotypically define and isolate rare HSCs. In the current issue of Cell Stem Cell, Wünsche et al. (2018) use γ-retroviral insertion sites from a human gene therapy trial to identify the active enhancer landscape of functionally characterized HSCs.