Isolation and <i>Ex Vivo</i>Expansion of Human Hematopoietic Stem Cells Derived from Umbilical Cord Blood

Umbilical cord blood (UCB) is a current major source of hematopoietic stem cells (HSCs) for cell transplantation therapy. Cell transplantation with HSCs derived from UCB is advantageous over transplantation with HSCs from adult tissues. However, the low number of HSC derived from a single unit of UCB limits its application. Thus, ex vivo expansion is a good option to create more UCB HSCs for clinical application. The strategies for HSC expansion in vitro focus on mimicking the composition and structure of HSC natural niche by enhancing self-renewal and inhibiting lineage differentiation of HSCs. In the past decade, the mechanisms of the interaction between HSC and the natural niche have been deeply investigated. This great progress in basic research has led to advancements in UCB HSC ex vivo expansion. In addition, the biological characteristics of the originally isolated UCB HSCs correlate with outcome of subsequent ex vivo expansion. In this paper, we summarize the late progress achieved in isolation and ex vivo expansion of UCB HSCs. Importantly, we attempt to provide an impact and practicable procedure to expand UCB HSC in vitro from isolation of original HSCs to identification of expanded HSCs.

The role of FZR1 in tumorigenesis: Focus on cell-cycle control

Fizzy-related protein homolog 1 (FZR1) mainly functions as a specific activator of the anaphase-promotingcomplex/cyclosome (APC/C) in the cell cycle and controls the G0 and G1 phases of the cell cycle. We highlightrecent work that has studied the role of FZR1 in tumorigenesis, growth, differentiation, and genome stability throughcell-cycle control. We summarize the current state of knowledge regarding FZR1 structure, function, and the distinctways of APC/C dysregulation in solid tumors and hematologic malignancies. We also discuss novel approaches fortargeting the FZR1 as a cancer therapy and research area for future work.

Construction and optimization of a base editor based on the MS2 system

Background:Catalytic defect Cas9‐cytosine deaminase fusion is widely used in base editing.The Multiple copy numbers of the MS2 binding site(MBS)can recruit multiple MS2 coat proteins(MCPs),which are usually applied to amplify signals.Our study aimed to apply the MS2 signal amplification system to the base editing system in order to achieve simultaneous mutations of multiple bases at the target genome site.Methods:Multiple copy numbers of the MS2 were ligated to the 3′‐end of sgRNA,and MCP was fused to the 5′‐end of cytosine deaminases.The MS2 was recognized by MCP to recruit cytosine deaminase for base substitutions(C‐T)at the target site.Different Cas9 variants,different cytosine deaminases and different copy numbers of MS2 were tested in this system,and the different versions of base editors were compared by editing efficiency and window.Results:In this study,dCas9,nCas9(D10A)and nCas9(H840A)were used.Among these 3 Cas9 variants,dCas9 exhibited higher base mutation efficiency.Two cytosine deaminases were then applied and the efficiency of rAPOBEC1 deaminase was found to be higher than AID.We also increased the copy numbers of MS2 linked to sgRNA from 2 to 12.Disappointingly,the sgRNA‐12x MS2 did not improve the editing efficiency or increase the editing window.Conclusion:An optimal version of base editor based on the MS2 system,MS2‐BErAPOBEC1(sgRNA‐2x MS2,MCP‐rAPOBEC1 and dCas9),was obtained.This tool can simultaneously mutate multiple bases at the target site,providing a new approach for the study of genome functions.

Overcoming adaptive resistance in AML by synergistically targeting FOXO3A-GNG7-mTOR axis with FOXO3A inhibitor Gardenoside and rapamycin

Therapeutic targeting FOxO3A(a forkhead transcription factor)represents a prom-ising strategy to suppress acute myeloid leukemia(AML).However,the effective inhibitors that target FOXO3A are lacking and the adaptive response signaling weakens the cytotoxic effect of FOxO3A depletion on AML cells.Here,we show that FOxO3A deficiency induces a compensa-tory response involved in the reactive activation of mTOR that leads to signaling rebound and adaptive resistance.Mitochondrial metabolism acts downstream of mTOR to provoke activa-tion of JNK/c-JUN via reactive oxygen species(ROS).At the molecular level,FOXO3A directly binds to the promoter of G protein gamma subunit 7(GNG7)and preserves its expression,while GNG7 interacts with mTOR and restricts phosphorylated activation of mTOR.Consequently,combinatorial inhibition of FOXO3A and mTOR show a synergistic cytotoxic effect on AML cells and prolongs survival in a mouse model of AML.Through a structure-based virtual screening,we report one potent small-molecule FOxO3A inhibitor(Gardenoside)that exhibits a strong effect of anti-FOXO3A DNA binding.Gardenoside synergizes with rapamycin to substantially reduce tumor burden and extend survival in AML patient-derived xenograft model.These results demonstrate that mTOR can mediate adaptive resistance to FOxO3A inhibition and validate a combinatorial approach for treating AML.

Immunoglobulin and granulocyte-colony stimulating factor affecting infection and hematopoietic reconstruction in allogeneic hematopoietic stem cell transplantation

To the Editor:Allogeneic hematopoietic stem cell transplantation(allo-HSCT)is an important treatment for hematologic disease.High-intensity preconditioning in allo-HSCT patients significantly increases the risk of severe infection,which is an important cause of early transplant-related death.

Clonal evolution analyses of a chronic myeloid leukemia patient with hematopoietic stem cell transplantation based on deep sequencing

Background The Philadelphia(Ph)chromosome is the hallmark chromosome aberration in chronic myeloid leukemia(CML),which confers the cancer phenotype of the disease.However,how the Ph chromosome forms and the genetic clonal evolution structure after targeted Ph treatment are still unclear.Methods In this study,we performed genome sequencing and clonal evolution analyses in a series of bone marrow specimens and skin biopsy from a CML patient who had received hematopoietic stem cell transplantation from her sister,then relapsed(lymphoid blast crisis),and received Ph-targeted therapy.Results The Ph chromosome was the“driver”clonal change in the original CML and the relapse.Both the patient and her sister had micro-deletions in the BCR gene region;however,the patient had a frameshift BRIP1 mutation that may account for the malfunctioning homologous recombination DNA repair of the BCR gene region and formation of the Ph chromosome.Conclusion We found that the BCR-ABL1 translocation was the driving force of the patient’s CML and relapse.The malfunctioning double-strand DNA break repair caused by the BRIP1 mutation could be the cause of Ph chromosome formation in the patient.

CACA guidelines for holistic integrative management of adult acute myeloid leukemia

The CACA Guidelines was summarized by Hematology Oncology Committee of China Anti-Cancer Association.This portion of the CACA Guidelines for adult acute myeloid leukemia(AML)not only focuses on diagnosis,the treat-ment options for younger(age<60 years)and older(age≥60 years)patients(including non-APL,APL,R/R AML),but also pay attention to the treatment of AML complications,including central nervous system leukemia(CNSL),cardiotoxicity,agranulocytosis and fever,hepatitis B virus reactivation,uric acid nephropathy,bleeding and coagula-tion disorders,and nursing for patients with AML from the perspective of holistic integrative medicine to enhance the quality of life and treatment effects.