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

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.

Correlation between Kir4.1 expression and barium-sensitive currents in rat and human glioma cell lines.

Gliomas are the most common primary brain tumors and often become apparent through symptomatic epileptic seizures. Glial cells express the inwardly rectifying K+ channel Kir4.1 playing a major role in K+ buffering, and are presumably involved in facilitating epileptic hyperexcitability. We therefore aimed to investigate the molecular and functional expression of Kir4.1 channels in cultured rat and human glioma cells. Quantitative PCR showed reduced expression of Kir4.1 in rat C6 and F98 cells as compared to control. In human U-87MG cells and in patient-derived low-passage glioblastoma cultures, Kir4.1 expression was also reduced as compared to autopsy controls. Testing Kir4.1 function using whole-cell patch-clamp experiments on rat C6 and two human low-passage glioblastoma cell lines (HROG38 and HROG05), we found a significantly depolarized resting membrane potential (RMP) in HROG05 (-29 ± 2 mV, n = 11) compared to C6 (-71 ± 1 mV, n = 12, P < 0.05) and HROG38 (-60 ± 2 mV, n = 12, P < 0.05). Sustained K+ inward or outward currents were sensitive to Ba2+ added to the bath solution in HROG38 and C6 cells, but not in HROG05 cells, consistent with RMP depolarization. While immunocytochemistry confirmed Kir4.1 in all three cell lines including HROG05, we found that aquaporin-4 and Kir5.1 were also significantly reduced suggesting that the Ba2+-sensitive K+ current is generally impaired in glioma tissue. In summary, we demonstrated that glioma cells differentially express functional inwardly rectifying K+ channels suggesting that impaired K+ buffering in cells lacking functional Ba2+-sensitive K+ currents may be a risk factor for increased excitability and thereby contribute to the differential epileptogenicity of gliomas.