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.

Development and characterization of a DNA aptamer for MLL-AF9 expressing acute myeloid leukemia cells using whole cell-SELEX

Current classes of cancer therapeutics have negative side effects stemming from off-target cytotoxicity. One way to avoid this would be to use a drug delivery system decorated with targeting moieties, such as an aptamer, if a targeted aptamer is available. In this study, aptamers were selected against acute myeloid leukemia (AML) cells expressing the MLL-AF9 oncogene through systematic evolution of ligands by exponential enrichment (SELEX). Twelve rounds of SELEX, including two counter selections against fibroblast cells, were completed. Aptamer pools were sequenced, and three candidate sequences were identified. These sequences consisted of two 23-base primer regions flanking a 30-base central domain. Binding studies were performed using flow cytometry, and the lead sequence had a binding constant of 37.5 + / − 2.5 nM to AML cells, while displaying no binding to fibroblast or umbilical cord blood cells at 200 nM. A truncation study of the lead sequence was done using nine shortened sequences, and showed the 5′ primer was not important for binding. The lead sequence was tested against seven AML patient cultures, and five cultures showed binding at 200 nM. In summary, a DNA aptamer specific to AML cells was developed and characterized for future drug-aptamer conjugates.

Preventing Brain Damage from Hypoxic–Ischemic Encephalopathy in Neonates: Update on Mesenchymal Stromal Cells and Umbilical Cord Blood Cells

Objective Neonatal hypoxic–ischemic encephalopathy (HIE) causes permanent motor deficit “cerebral palsy (CP),” and may result in significant disability and death. Therapeutic hypothermia (TH) had been established as the first effective therapy for neonates with HIE; however, TH must be initiated within the first 6 hours after birth, and the number needed to treat is from 9 to 11 to prevent brain damage from HIE. Therefore, additional therapies for HIE are highly needed. In this review, we provide an introduction on the mechanisms of HIE cascade and how TH and cell therapies such as umbilical cord blood cells and mesenchymal stromal cells (MSCs), especially umbilical cord-derived MSCs (UC-MSCs), may protect the brain in newborns, and discuss recent progress in regenerative therapies using UC-MSCs for neurological disorders. Results The brain damage process “HIE cascade” was divided into six stages: (1) energy depletion, (2) impairment of microglia, (3) inflammation, (4) excitotoxity, (5) oxidative stress, and (6) apoptosis in capillary, glia, synapse and/or neuron. The authors showed recent 13 clinical trials using UC-MSCs for neurological disorders. Conclusion The authors suggest that the next step will include reaching a consensus on cell therapies for HIE and establishment of effective protocols for cell therapy for HIE. Key Points

Femoral Heads from Total Hip Arthroplasty as a Source of Adult Hematopoietic Cells

Normal human bone marrow cells are critical for studies of hematopoiesis and as controls to assess toxicity. As cells from commercial vendors are expensive, many laboratories resort to cancer-free bone marrow specimens obtained during staging or to umbilical cord blood cells, which may be abnormal or reflect a much younger age group compared to the disease samples under study. We piloted the use of femoral heads as an alternative and inexpensive source of normal bone marrow. Femoral heads were obtained from 21 successive patients undergoing elective hip arthroplasty. Mononuclear cells (MNCs) were purified with Ficoll, and CD3⁺, CD14⁺, and CD34⁺ cells were purified with antibody-coated microbeads. The median yield of MNCs was 8.95 × 10⁷ (range, 1.62 × 10⁵–2.52 × 10⁸), and the median yield of CD34⁺ cells was 1.40 × 10⁶ (range, 3.60 × 10⁵–9.90 × 10⁶). Results of downstream applications including qRT-PCR, colony-forming assays, and ex vivo proliferation analysis were of high quality and comparable to those obtained with standard bone marrow aspirates. We conclude that femoral heads currently discarded as medical waste are a cost-efficient source of bone marrow cells for research use.