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.

Clinical Practice of Umbilical Cord Blood Stem Cells in Transplantation and Regenerative Medicine - Prodigious Promise for Imminent Times

: The umbilical cord blood is usually disposed of as an unwanted material after parturition; however, today, it is viewed as a regenerative medication so as to create the organ tissues. This cord blood gathered from the umbilical cord is made up of mesenchymal stem cells, hematopoietic stem cells, and multipotent non-hematopoietic stem cells having many therapeutic effects as these stem cells are utilized to treat malignancies, hematological ailments, inborn metabolic problem, and immune deficiencies. Presently, numerous clinical applications for human umbilical cord blood inferred stem cells, as stem cell treatment initiate new research. These cells are showing such a boon to stem cell treatment; it is nevertheless characteristic that the prospect of conservation of umbilical cord blood is gaining impetus. Current research works have demonstrated that about 80 diseases, including cancer, can be treated or relieved utilizing umbilical cord blood stem cells, and every year, many transplants have been effectively done around the world. However, in terms of factors, including patient selection, cell preparation, dosing, and delivery process, the treatment procedure for therapy with minimally manipulated stem cells can be patented. It is also worth thinking about how this patent could affect cord blood banks. Meanwhile, the utilization of cord blood cells is controversial and adult-derived cells may not be as successful, so numerous clinicians have begun working with stem cells that are acquired from umbilical cord blood. This review epitomizes a change in outlook from what has been completed with umbilical cord blood cell research and cord blood banking on the grounds that cord blood cells do not require much in the method of handling for cryopreservation or for transplantation in regenerative medicine.

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.

Use of Modern Cryomethods in Combined Surgical Treatment of Patients with Purulent Chronic Wounds

The paper considers the possibilities of using in medical practice various cryomethods and techniques of cryopreservation of biological objects to treat the patients with purulent chronic wounds (PCW). The variety of methods and approaches to apply cold in medical practice: general and local cryotherapy, cryosurgery etc. has been emphasized. As a result of using each of these methods, regenerative processes begin, which promote the healing of PCW. Particular attention is paid to cryotechniques, ensuring the preservation of cord blood at low temperatures. Cord blood cells and serum have been shown to have immune modulatory and trophic-stimulating therapeutic effects, that is extremely important when treating the patients with PCW. The issue of combined use of cord blood serum and innovative vacuum therapy for the patients with PCW has been considered.

Potential HIV gene therapy strategies

The CCR5 null genotype generation has been a main focus in the HIV gene therapy industry. The presence of the X4 tropic virus, mobilization of HSPCs, the quality of the cells for manipulation, and gene editing efficiency appear to be the main obstacles in translating this technique. Unintended off-target cleavage is a key problem in CRISPR/Cas9 editing. With the development of small molecule expansion methods for cord blood HSPC, it would be advantageous to modify CCR5 in cord blood cells and expand them for transplantation. The generation of engraftable HSPCS from iPSCs would be an ideal technique for HSCC gene therapy.The haplotype-characterized iPSC would be the donor for many patients, and it could be a commercially available product. The 32 C CR5 homozygous people had no elevated mortality risks according to whole-exome sequencing and whole-genome genotyping, according to CCR 5 positive people, and had no higher mortality risks compared to those who were HIV positive. Recent advances in gene editing, such as non-viral delivery of Cas9 ribonucleoproteins, incorporation of a 3X-nuclear localization signal into spCas9, and use of HiFi Cas9 with chemically modified sgRNAs, can be combined with recent advances in transplantation. Infusing modest doses of gene modified primitive HSPC fractions indicated by CD34 + CD90 + CD45RA-, which can engraft better, is another option for lowering the cost of gene therapy.

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

Reduced angiovasculogenic and increased inflammatory profiles of cord blood cells in severe but not mild preeclampsia

Preeclampsia (PE) is a prevalent pregnancy disorder that leads to high maternal and fetal morbidity and mortality. While defective vascular development and angiogenesis in placenta are known as crucial pathological findings, its pathophysiological mechanism remains elusive. To better understand the effects of PE on angio-vasculogenesis and inflammatory networks in the fetus and to identify their biological signatures, we investigated the quantitative and functional characteristics of cord blood-derived mononuclear cells (CB-MNCs) and CD31-positive MNCs. Flow cytometry analysis demonstrated that the CB-MNCs from the severe PE group had significantly decreased number of cells expressing CD3, CD11b, CD14, CD19, KDR, and CD31 compared with the normal group. Quantitative real time PCR (qRT-PCR) shows down-regulation of the major angiogenic factor VEGFA in MNCs and CD31+ MNCs in severe PE. The major inflammatory cytokines IL1 was highly upregulated in CD31+ CB-MNCs in the severe PE patients. Mild PE patients, however, did not display any significant difference in expression of all measured angiogenic genes and most inflammatory genes. These findings show distinct angiogenic and inflammatory signatures from severe PE, and they may play a significant role in the pathogenesis of vascular defects in placenta of severe PE.