Abstract
To investigate clonal hematopoiesis associated gene mutations in vitro and to unravel the direct impact on the human stem and progenitor cell (HSPC) compartment, we targeted healthy, young hematopoietic progenitor cells, derived from umbilical cord blood samples, with CRISPR/Cas9 technology. Site-specific mutations were introduced in defined regions of DNMT3A, TET2, and ASXL1 in CD34+ progenitor cells that were subsequently analyzed in short-term as well as long-term in vitro culture assays to assess self-renewal and differentiation capacities. Colony-forming unit (CFU) assays revealed enhanced self-renewal of TET2 mutated (TET2mut) cells, whereas ASXL1mut as well as DNMT3Amut cells did not reveal significant changes in short-term culture. Strikingly, enhanced colony formation could be detected in long-term culture experiments in all mutants, indicating increased self-renewal capacities. While we could also demonstrate preferential clonal expansion of distinct cell clones for all mutants, the clonal composition after long-term culture revealed a mutation-specific impact on HSPCs. Thus, by using primary umbilical cord blood cells, we were able to investigate epigenetic driver mutations without confounding factors like age or a complex mutational landscape, and our findings provide evidence for a direct impact of clonal hematopoiesis-associated mutations on self-renewal and clonal composition of human stem and progenitor cells.
Highlights
Hematopoietic stem and progenitor cells (HSPCs) are continuously generating mature blood cells
CRISPR/Cas9 modeling in primary human HSPCs affects TET2 protein function Mononuclear cells were isolated from freshly drawn umbilical cord blood (CB)
The cells were transfected with RNPs targeting ASXL1 exon 13, TET2 exon 6, or DNMT3A exon 23 plus the homology directed repair (HDR) templates to introduce the mutation R882H in DNMT3A exon 23 or a 8 bp deletion in ASXL1 exon 13
Summary
Hematopoietic stem and progenitor cells (HSPCs) are continuously generating mature blood cells. Some mutations might confer a proliferative and/or self-renewal advantage to the HSPCs, leading to preferential expansion of specific clone(s) This represents an effect mainly seen in the aging hematopoietic system. Large sequencing studies have identified recurrent mutations in blood cells of elderly individuals suffering from non-hematologic diseases [2, 3] or healthy elderly individuals [3, 4] This phenomenon, termed clonal hematopoiesis of indeterminate potential (CHIP), is defined by the presence of a hematologic malignancy-associated gene mutation with a variant allele frequency (VAF) of at least 2% [5]. Several groups have demonstrated that similar mutations can be found in a pre-leukemic cell population in patients with full-blown acute leukemia These clones may survive chemotherapy, persist during remission, and have a fitness advantage over normal HSCs [9,10,11,12]
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