Modeling clonal hematopoiesis in umbilical cord blood cells by CRISPR/Cas9

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.

Clonal hematopoiesis and cardiovascular disease: deciphering interconnections

“…The exact mechanisms by which some mutations promote a clonal advantage and accelerate atherosclerosis, for instance by provoking inflammation, remain to be elucidated. From what is known so far, CHIP mutations convey enhanced self-renewal of the hematopoietic stem cell (HSC) compartment and concomitantly obstructed hematopoietic differentiation in a mutation-specific fashion [ 24 , 25 , 47 ]. The most frequent mutations within TET2 and DNMT3A further promote granulomonocytic differentiation to the expense of the erythroid lineage [ 57 , 88 ].…”

Clonal Hematopoiesis: Role in Hematologic Non-Hematologic

“…Interestingly, a recent study showed an experimental approach based on CRISPR/Cas9 technology to develop a simple model of clonal hematopoiesis. 133 Site-specific mutations were introduced in specific sites of ASXL1, DNMT3A , and TET2 in CD34 + progenitors derived from umbilical cord blood. The biological effects induced by these genetic modifications were assayed in short-term and long-term cultures, evaluating changes in self-renewal and cell differentiation; TET2 , but not DNMT3A and ASXL1 mutations induced enhanced self-renewal in short-term cultures; all the three mutants and particularly the combined three mutants elicited a clear increase of self-renewal, as evidenced by long-term culture experiments.…”

“… 133 In addition, the analysis of clonal expansion after long-term culture showed a mutation-specific impact on stem/progenitor cells. 133 …”

“…The biological effects induced by these genetic modifications were assayed in short-term and long-term cultures, evaluating changes in selfrenewal and cell differentiation; TET2, but not DNMT3A and ASXL1 mutations induced enhanced self-renewal in short-term cultures; all the three mutants and particularly the combined three mutants elicited a clear increase of self-renewal, as evidenced by long-term culture experiments. 133 In addition, the analysis of clonal expansion after long-term culture showed a mutation-specific impact on stem/progenitor cells. 133 The study of long-term survivors of allogeneic stem cell transplantation grafted with CHIP-positive donors offers the unique opportunity to explore the expansion of CHIP clones during hematopoietic reconstitution.…”

“…133 In addition, the analysis of clonal expansion after long-term culture showed a mutation-specific impact on stem/progenitor cells. 133 The study of long-term survivors of allogeneic stem cell transplantation grafted with CHIP-positive donors offers the unique opportunity to explore the expansion of CHIP clones during hematopoietic reconstitution. Boettcher et al have studied 5 of these patients exhibiting donor-engrafted CHIP: 4/5 cases displayed increased CHIP clones' size in recipients compared with donors, as measured by VAF; CHIP mutations were constantly found in the myeloid lineage, but with variable penetrance in the B and T lymphoid lineages; telomere shortening was observed in granulocytes, supporting a proliferative activity of hematopoietic stem cells.…”