Lymphoid-biased hematopoietic stem cells and myeloid-biased hematopoietic progenitor cells have radioprotection activity

Radioprotection was previously considered as a function of hematopoietic stem cells(HSCs).However,recent studies have reported its activity in hematopoietic progenitor cells(HPCs).To address this issue,we compared the radioprotection activity in 2 subsets of HSCs(nHSC1 and 2 populations)and 4 subsets of HPCs(nHPC1–4 populations)of the mouse bone marrow,in relation to their in vitro and in vivo colony-forming activity.Significant radioprotection activity was detected in the nHSC2 population enriched in lymphoid-biased HSCs.Moderate radioprotection activity was detected in nHPC1 and 2 populations enriched in myeloid-biased HPCs.Low radioprotection activity was detected in the nHSC1 enriched in myeloid-biased HSCs.No radioprotection activity was detected in the nHPC3 and 4 populations that included MPP4(LMPP).Single-cell colony assay combined with flow cytometry analysis showed that the nHSC1,nHSC2,nHPC1,and nHPC2 populations had the neutrophils/macrophages/erythroblasts/megakaryocytes(nmEMk)differentiation potential whereas the nHPC3 and 4 populations had only the nm differentiation potential.Varying day 12 spleen colony-forming units(day 12 CFU-S)were detected in the nHSC1,nHSC2,and nHPC1–3 populations,but very few in the nHPC4 population.These data suggested that nmEMk differentiation potential and day 12 CFU-S activity are partially associated with radioprotection activity.Reconstitution analysis showed that sufficient myeloid reconstitution around 12 to 14 days after transplantation was critical for radioprotection.This study implied that radioprotection is specific to neither HSC nor HPC populations,and that lymphoid-biased HSCs and myeloid-biased HPCs as populations play a major role in radioprotection.

Chemoprotection of transfer of multidrug resistance gene into human hematopoietic progenitor cell

Objective To observe the effect of the transfer of multidrug resistance gene (mdr1) into human hematopoietic progenitor cells (HPC) on the chemoprotection Methods Human CD34 + cells served as a target of mdr1 gene transfer Retroviral vector SF mdr containing human total length mdr1cDNA was introduced into packing cells GP envAM12 by liposome mediated transfection The mdr1 gene was transduced into human CD34 + cells by retroviral supernatants of packing cells The integration and expression of the mdr1 gene and its protein (P170) in transduced cells were determined by PCR, RT PCR, and flow cytometry The drug resistance of chemotherapy in transduced HPC was determined by culturing colonies Results The mdr1 gene was integrated and expressed in transduced CD34 + cells The efficiency of mdr1 gene transfer was 10%-14% Compared with untransduced controls, within a certain range of drug concentration, the number of drug resistant colony in transduced HPC for taxol, doxorubicin,VCR and VP16 were increased by 3 6±2 1 fold, 2 9±0 3 fold, 1 9±0 4 fold, and 3 5±0 5 fold, respectively Conclusion The transfer of the mdr1 gene into human HPC can increase the drug resistance of the transduced cells to corresponding chemotherapeutic drugs that may provide some degree of chemoprotection for HPC

Inducing effects of macrophage stimulating protein on the expansion of early hematopoietic progenitor cells in liquid culture

Background Macrophage stimulating protein （MSP） is produced by human bone marrow endothelial cells. In this study we sought to observe its effects on inducing the expansion of early hematopoietic progenitor cells which were cultured in a liquid culture system in the presence of the combination of stem cell factor （SCF）, interleukin 3 （IL-3）, interleukin 6 （IL-6）, granulocyte macrophage-colony stimulating factor （GM-CSF）, erythropoietin （EPO） （Cys） and MSP or of Cys and bone marrow endothelial cell conditioned medium （EC-CM）. Methods Human bone marrow CD34^＋ cells were separated and cultured in a liquid culture system for 6 days. Granulocyte-macrophage colony forming unit （CFU-GM） and colony forming unit-granulocyte, erythrocyte, macrophage, megakaryocyte （CFU-GEMM） were employed to assay the effects of different treatment on the proliferation of hematopoeitic stem/progenitor cells. The nitroblue tetrazolium （NBT） reductive test and hoechest 33258 staining were employed to reflect the differentiation and apoptosis of the cells respectively. Results MSP inhibited the proliferation of CFU-GM and CFU-GEMM in semi-solid culture and the inhibitory effect on CFU-GEMM was stronger than on CFU-GM. MSP inhibited the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators. Bone marrow （BM） CFU-GEMM was 2.3-fold or 1.7-fold increase or significantly decreased in either Cys＋EC-CM, Cys＋MSP or Cys compared with 0 hour control in liquid culture system after 6 days. Conclusion MSP, a hematopoietic inhibitor, inhibits the differentiation of early hematopoietic progenitor cells induced by hematopoietic stimulators and makes the early hematopoietic progenitor cells expand in a liquid culture system.

The Effects of Total Saponins of Panax Ginseng on Hematopoietic Progenitor Cells in Healthy Humans and Aplastic Anemia Patients

Ginseng is said to have beneficial effects on anemia. The proliferation effects of totalsaponins of Panax ginseng (TSPG) on hematopoietic progenitor cell in healthy individuals and 29 patientswith aplastic anemia (AA) were observed through bone marrow cultures of burst forming unit-erythroid(BFU-E) , colony forming unit-erythroid (CFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) in vitrcacompared with methyltestosterone (MT). The results suggest TSPG might prompt the prolif-eration of normal progenitor cellS at a concentration of 20 g/ml. The numbers of BFU-E ,CFU-E and CFU-GM increased by 37. 8±2.9 % , 31. 4±2. 9 % and 33. 3± 4. 0 % respectively over the controls ; further-more TSPG was still useful to BFU-E,CFU-E growth without Epo in vitro, although the colony nurnberswere much lower. Otherwise MT was useless to CFUGM. Of the 29 patients with AA, 14 who respondedto MT showed sensitivity to TSPG in marrow culture (the rising rate of colony formation exceeded 30 % ) ,but immune-mediated AA (patient's peripheral blood mononucleated cell suppressed normalhematopoiesis) and stem cell decreased AA (few of colonies were formed) showed almost no expressionfor TSPG activity because of the immunological suppression system and the absence of progenitors.

Establishment of a humanized mouse model using steady-state peripheral blood-derived hematopoietic stem and progenitor cells facilitates screening of cancer-targeted T-cell repertoires

Background:Cancer-targeted T-cell receptor T(TCR-T)cells hold promise in treating cancers such as hematological malignancies and breast cancers.However,approaches to obtain cancer-reactive TCR-T cells have been unsuccessful.Methods:Here,we developed a novel strategy to screen for cancer-targeted TCR-T cells using a special humanized mouse model with person-specific immune fingerprints.Rare steady-state circulating hematopoietic stem and progenitor cells were expanded via three-dimensional culture of steady-state peripheral blood mononuclear cells,and then the expanded cells were applied to establish humanized mice.The human immune system was evaluated according to the kinetics of dendritic cells,monocytes,T-cell subsets,and cytokines.To fully stimulate the immune response and to obtain B-cell precursor NAML-6-and triple-negative breast cancer MDA-MB-231-targeted TCR-T cells,we used the inactivated cells above to treat humanized mice twice a day every 7 days.Then,human T cells were processed for TCRβ-chain(TRB)sequencing analysis.After the repertoires had been constructed,features such as the fraction,diversity,and immune signature were investigated.Results:The results demonstrated an increase in diversity and clonality of T cells after treatment.The preferential usage and features of TRBV,TRBJ,and the V–J combination were also changed.The stress also induced highly clonal Science and Technology,Grant/Award Number:2021C03010;Zhejiang Provincial Natural Science Foundation of China,Grant/Award Numbers:LTGY24H080003,LY21H080004 expansion.Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice.Conclusions:We constructed a personalized humanized mouse model to screen cancer-targeted TCR-T pools.Our platform provides an effective source of cancer-targeted TCR-T cells and allows for the design of patient-specific engineered T cells.It therefore has the potential to greatly benefit cancer treatment.

Liposome-mediated Functional Expression of Multiple Drug Resistance Gene in Human Bone Marrow CD34^+ Cells

Summary: The expression and functional activity of multiple drug resistance (MDR1) gene in human normal bone marrow CD34+ cells was observed. Human normal bone marrow CD34+ cells were enriched with magnetic cell sorting (MACS) system, and then liposome-mediated MDR1 gene was transferred into bone marrow CD34+ cells. Fluorescence-activated cell sorter was used to evaluate the expression and functional activity of P-glycoprotein (P-gp) encoded by MDR1 gene. It was found that the purity of bone marrow CD34+ cells was approximately (91±4.56) % and recovery rate was (72.3±2.36) % by MACS. The expression of P-gp in the transfected CD34+cells was obviously higher than that in non-transfected CD34+ cells. The amount of P-gp in non-transfected CD34+ cells was (11.2±2.2) %, but increased to (23.6±2.34) % 48 h after gene transfection (P<0.0l). The amount of P-gp was gradually decreased to the basic level one week later. The accumulation and extrusion assays showed that the overexpression of P-gp could efflux Rh-123 out of cells and there was low fluorescence within the transfected cells. The functional activity of P-gp could be inhibited by 10 μg/ml verapamil. It was suggested that the transient and highly effective expression and functional activity of P-gp could be obtained by liposome-mediated MRD1 transferring into human normal bone marrow CD34+ cells.

Exendin-4 attenuates atherosclerosis progression via controlling hematopoietic stem/progenitor cell proliferation

Beyond glycemic control, applications of glucagon-like peptide-1 receptor (GLP-1r) agonists (GLP-1 RAs) inhibit inflammationand plaque development in murine atherosclerotic models. However, whether they modulate hematopoietic stem/progenitor cells(HSPCs)to prohibit skewed myelopoiesis in hypercholesteremia remains unknown. In this study, GLP-1r expression in fluorescenceactivated cell sorting (FACS)-sorted wild-type HSPCs was determined by capillary western blotting. Bone marrow cells (BMCs)of wild-type or GLP-1r−/− mice were transplanted into lethally irradiated low-density lipoprotein receptor deficient (LDLr−/−)recipients followed by high-fat diet (HFD) for chimerism analysis by FACS. In parallel, LDLr−/− mice were placed on HFD for 6weeks and then treated with saline or Exendin-4 (Ex-4) for another 6 weeks. HSPC frequency and cell cycle were analyzed byFACS, and intracellular metabolite levels were assessed by targeted metabolomics. The results demonstrated that HSPCs expressedGLP-1r and transplantation of GLP-1r−/− BMCs resulted in skewed myelopoiesis in hypercholesterolemic LDLr−/− recipients.In vitro, Ex-4 treatment of FACS-purified HSPCs suppressed cell expansion and granulocyte production induced by LDL. In vivo, Ex-4treatment inhibited plaque progression, suppressed HSPC proliferation, and modified glycolytic and lipid metabolism in HSPCs ofhypercholesteremic LDLr−/− mice. In conclusion, Ex-4 could directly inhibit HSPC proliferation induced by hypercholesteremia.

Effect of Angiotensin Ⅱ on Cord Blood CD^(34+) Cells Expansion in vitro

In order to investigate the influence of angiotensin Ⅱ on hematopoietic system, CD34 + cells in cord blood were purified, and the effects of angiotensin Ⅱ in combination with various cytokines on their growth and differentiation were studied by cell culture in vitro. It was found that angiotensin Ⅱ in suspending medium could stimulate both BFU-E and CFU-GM expansion. The number of BFU-E and CFU-GM was increased with the increases of angiotensin Ⅱ concentrations during a certain range. In addition, the expansion fold of CFU-GM was increased from 2.3±0.8 times to 7.8±2.3 times when angiotensin Ⅱ was added in the presence of SCF+G-CSF+GM-CSF+IL-3 cytokines mixture. Similarly, the expansion fold of BFU-E was increased from 3.1±1.8 times to 9 2±2.3 times with angiotensin Ⅱ in the presence of SCF+EPO+TPO+IL-3. In the semi-solid medium, angiotensin Ⅱ could stimulate CFU-GM expansion but had no effect on the growth of BFU-E. In conclusion, angiotensin Ⅱ had some stimulating effects on cord blood hematopoietic progenitors expansion in vitro in the presence of other cytokines.

Efficient expansion of rare human circulating hematopoietic stem/progenitor cells in steady-state blood using a polypeptide-forming 3D culture

Although widely applied in treating hematopoietic malignancies,transplantation of hematopoietic stem/progenitor cells(HSPCs)is impeded by HSPC shortage.Whether circulating HSPCs(cHSPCs)in steady-state blood could be used as an alternative source remains largely elusive.Here we develop a three-dimensional culture system(3DCS)including arginine,glycine,aspartate,and a series of factors.Fourteen-day culture of peripheral blood mononuclear cells(PBMNCs)in 3DCS led to 125-and 70-fold increase of the frequency and number of CD34+cells.Further,3DCS-expanded cHSPCs exhibited the similar reconstitution rate com-pared to CD34+HSPCs in bone marrow.Mechanistically,3DCS fabricated an immunomodulatory niche,secreting cytokines as TNF to support cHSPC survival and proliferation.Finally,3DCS could also promote the expansion of cHSPCs in patients who failed in HSPC mobilization.Our 3DCS successfully expands rare cHSPCs,providing an alternative source for the HSPC therapy,particularly for the patients/donors who have failed in HSPC mobilization.

RNA methylation regulates hematopoietic stem and progenitor cell development

Methylation of adenosine base on the nitrogen-6 position （N6-methyladenosine, m^6A） is the most common and abundant modification on mRNA transcripts. This post-transcriptional modification was first described in the 1970s in hepatoma cells （Desrosiers et al., 1974）.

3D collagen matrices modulate the transcriptional trajectory of bone marrow hematopoietic progenitors into macrophage lineage commitment

Physical signals provided by the extracellular matrix(ECM)are key microenvironmental parameters for the fate decision of hematopoietic stem and progenitor cells(HSPC)in bone marrow.Insights into cell-ECM interactions are critical for advancing HSC-based tissue engineering.Herein,we employed collagen hydrogels and collagen-alginate hydrogels of defined stiffness to study the behaviors of hematopoietic progenitor cells(HPCs).Three-dimensional(3D)collagen hydrogels with a stiffness of 45 Pa were found to promote HPC maintenance and colony formation of monocyte/macrophage progenitors.Using single-cell RNA sequencing(scRNA-seq),we also characterized the comprehensive transcriptional profiles of cells randomly selected from two-dimensional(2D)and 3D hydrogels.A distinct maturation trajectory from HPCs into macrophages within the 3D microenvironment was revealed by these results.3D-derived macrophages expressed high levels of various cytokines and chemokines,such as Saa3,Cxcl2,Socs3 and Tnf.Furthermore,enhanced communication between 3D-macrophages and other hematopoietic clusters based on ligand-repair interactions was demonstrated through bioinformatic analyses.Our research underlines the regulatory role of matrix-dimensionality in HPC differentiation and therefore probably be applied to the generation of specialized macrophages.

Experimental study on ex vivo expanded hematopoietic stem/progenitor in the two step culture from human umbilical cord blood transplanted into NOD/SCID mice

The effects of hematopoietic stem/progenitor cells(HSPCs)expanded in the two step coculture with human bone marrow mesenchymal stem cells(hMSCs)on the hematopoietic reconstruction of irradiated NOD/SCID mice were studied.Mononuclear cells(MNCs)were isolated from human umbilical cord blood(UCB)and cultured in the non-coculture scheme of rhSCF+rhG−CSF+rhMDGF combination and the coculture scheme of rhSCF+rhG−CSF+rhMDGF+hMSCs.Sublethally-irradiated NOD/SCID mice were transplanted with ex vivo expanded HSPCs with the dose of 8.5×10^(6) cells per mouse.After transplantation,the dynamics of WBC in the transplanted mice was measured periodically,and the Alu sequence fragment special for human in the transplanted mice was inspected by PCR.Results showed that the coculture scheme increased proliferation of UCB-derived HSPCs.After transplantation with expanded HSPCs,the population of WBC in the transplanted mice increased in 12 d and reached the first peak in 25 d,then showed the second increasing of WBC in 45~55 d.Expanded cells from the coculture scheme appeared to be favorable for the second increasing of WBC in the transplanted mice.After 85 d,the Alu sequence fragment was detected in the probability of 87.5%(7/8)for the non-coculture scheme and 88.9%(8/9)for the coculture scheme.

Stimulating effect of catechin, an active component of Spatholobus suberectus Dunn, on bioactivity of hematopoietic growth factor

Background Hematopoietic growth factor （HGF） is indispensable to hematopoiesis in the body. The proliferation and differentiation of hematopoietic cells must rely on the existence and stimulation of HGF. This study investigated the effect of catechin, an active component extracted from Spatholobus suberectus Dunn （SSD）, on bioactivity of granulocyte-macrophage colony-stimulating activity （GM-CSA）, burst-promoting activity （BPA） and megakaryocyte colony-stimulating activity （MK-CSA） in spleen condition medium （SPCM） of mice to clarify the hematopoietic mechanism of catechin and SSD. Methods Spleen cells of mice were separated and spleen condition medium （SPCM） was prepared from spleen cell culture. Bone marrow cells of mice were separated and cultured in a culture system including 10% （v/v） SPCM （induced by catechin in vivo or ex vivo） for 6 days. Granulocyte-macrophage colony forming units （CFU-GM）, erythrocyte burst-colony-forming units （BFU-E） and megakaryocyte colony-forming units （CFU-Meg） formation were employed to assay the effects of different treatment on the bioactivity of GM-CSA, BPA and MK-CSA in SPCM. Results SPCM induced by 100 mg/L catechin ex vivo could promote the growth of CFU-GM, BFU-E and CFU-Meg, which indicated that catechin could stimulate the production of GM-CSA, BPA and MK-CSA in SPCM. SPCM prepared at the fourth day of spleen cell culture showed the best stimulating activity. The bioactivity of GM-CSA, BPA and MK-CSA in the SPCM prepared after intraperitoneally injecting catechin into mice was also increased. The number of CFU-GM, BFU-E and CFU-Meg gradually increased as the dose of catechin increased and the time of administration prolonged. CFU-GM, BFU-E and CFU-Meg of the high-dose catechin group were significantly higher than those of the control group （P〈0.01） and reached the maximum at the seventh day after administration. Conclusions This study suggests that catechin extracted from the active acetic ether part of Spatholobus suberectus Dunn can regulate hematopoiesis by inducing bioactivity of GM-CSA, BPA and MK-CSA in SPCM of mice. This may be one of the mechanisms for the hematopoietic-supportive effect of catechin and Spatholobus suberectus Dunn.

The evolving views of hematopoiesis:from embryo to adulthood and from in vivo to in vitro

The hematopoietic system composed of hematopoietic stem and progenitor cells(HSPCs)and their differentiated lineages serves as an ideal model to uncover generic principles of cell fate transitions.From gastrulation onwards,there successively emerge primitive hematopoiesis(that produces specialized he-matopoietic cells),pro-definitive hematopoiesis(that produces lineage-restricted progenitor cells),and definitive hematopoiesis(that produces multipotent HSPCs).These nascent lineages develop in several transient hematopoietic sites and finally colonize into lifelong hematopoietic sites.The development and maintenance of hematopoietic lineages are orchestrated by cell-intrinsic gene regulatory networks and cell-extrinsic microenvironmental cues.Owing to the progressive methodology(e.g.,high-throughput lineage tracing and single-cell functional and omics analyses),our understanding of the developmental origin of hematopoietic lineages and functional properties of certain hematopoietic organs has been updated;meanwhile,new paradigms to characterize rare cell types,cell heterogeneity and its causes,and comprehensive regulatory landscapes have been provided.Here,we review the evolving views of HSPC biology during developmental and postnatal hematopoiesis.Moreover,we discuss recent advances in the in vitro induction and expansion of HSPCs,with a focus on the implications for clinical applications.

Hematopoietic-supportive effect of (2S, 3R)-ent-catechin on marrow-depressed mice

Hematopoiesis is an active process of cell proliferation, differentiation andrelease. It is the process during which hematopoietic stem cells (HSCs) proliferate anddifferentiate to mature blood cells under the effect of hemetopoietic growth factors (HGFs) incertain hematopoietic microenvironment. HSCs are sources of hematopoiesis of a body that canself-renew, differentiate to blood cells of every lineage and maintain the constancy of them. As themajor tissue of hematopoiesis bone marrow is filled with all kinds of blood cells in variousdevelopmental stages. Under the normal conditions, the ordered proliferation and differentiation ofhematopoietic stem cells/hematopoietic progenitor cells ( HSC/HPC) depend on the regulation ofcytokine network. In present, Spatholobus suberectus Dunn (SSD), a traditional Chinese herb medicinethat has been used to invigorate the blood circulation for thousands years, is widely used torebuild blood after chemotherapy and radiotherapy and to treat hematopoietic diseases. Previousreports from our laboratory indicated that (2S,3R)-ent-catechin, epi (2S, 3R )-ent-catechin, gallic( 2S, 3R )-ent-catechin, et al extracted from SSD all could stimulate the proliferation of HPC inmarrow depressed mice, among which (2S, 3R)-ent-catechin obtained from spatholobus genus for thefirst time and whose content was the maxim showed the best effect. In this study, firstly, theeffect of (2S,3R)-ent-catechin on the quality and quantity of HSCs were assayed by detectingCD_(34)^+ expression. Secondly, we investigated the variation of cytokine (GM-CSF and IL-6)sero-level in mice treated with (2S,3R)- ent-catechin by ELISA. And IL-6 mRNA and GM-CSF mRNAexpressions induced by (2S,3R)-ent-catechin were detected simultaneously by RT-PCR technique inorder to clarify its mechanism.

Two-step protocol for regeneration of immunocompetent T cells from mouse pluripotent stem cells

Numerous efforts have been attempted to regenerate T cells in culture dish from pluripotent stem cells(PSCs).However,in vitro generated T cells exhibited extremely low activity and compromised immunocompetency in vivo.Here,we describe a two-step protocol for regenerating functional T cells using an inducible Runx1-Hoxa9-PSC(iR9-PSCs)line.The procedure mainly includes generation of induced hematopoietic progenitor cells(iHPCs)in vitro,transplantation,and development of functional induced T cells(iT)in vivo via transplantation.The entire induction process in vitro requires 21 days before iHPCs transplantation.The development of mature T cells in vivo takes 4 to 6 weeks post-transplantation.We provide a simple and reproducible approach for functional T cell regeneration from iR9-PSCs for research purpose.

Adult-repopulating lymphoid potential of yolk sac blood vessels is not confined to arterial endothelial cells

During embryogenesis,hematopoietic stem progenitor cells(HSPCs)are believed to be derived from hemogenic endothelial cells(HECs).Moreover,arterial feature is proposed to be a prerequisite for HECs to generate HSPCs with lymphoid potential.Although the molecular basis of hematopoietic stem cell-competent HECs has been delicately elucidated within the embryo proper,the functional and molecular characteristics of HECs in the extraembryonic yolk sac(YS)remain largely unresolved.In this study,we initially identified six molecularly different endothelial populations in the midgestational YS through integrated analysis of several single-cell RNA sequencing(scRNA-seq)datasets and validated the arterial vasculature distribution of Gja5+ECs using a Gja5-EGFP reporter mouse model.Further,we explored the hemogenic potential of different EC populations based on their Gja5-EGFP and CD44 expression levels.The hemogenic potential was ubiquitously detected in spatiotemporally different vascular beds on embryonic days(E)8.5–E9.5 and gradually concentrated in CD44-positive ECs from E10.0.Unexpectedly,B-lymphoid potential was detected in the YS ECs as early as E8.5 regardless of their arterial features.Furthermore,the capacity for generating hematopoietic progenitors with in vivo lymphoid potential was found in nonarterial as well as arterial YS ECs on E10.0–E10.5.Importantly,the distinct identities of E10.0–E10.5 HECs between YS and intraembryonic caudal region were revealed by further scRNA-seq analysis.Cumulatively,these findings extend our knowledge regarding the hemogenic potential of ECs from anatomically and molecularly different vascular beds,providing a theoretical basis for better understanding the sources of HSPCs during mammalian development.

A DL-4- and TNFα-based culture system to generate high numbers of nonmodified or genetically modified immunotherapeutic human T-lymphoid progenitors

Several obstacles to the production,expansion and genetic modification of immunotherapeutic T cells in vitro have restricted the widespread use of T-cell immunotherapy.In the context of HSCT,delayed naïve T-cell recovery contributes to poor outcomes.A novel approach to overcome the major limitations of both T-cell immunotherapy and HSCT would be to transplant human T-lymphoid progenitors(HTLPs),allowing reconstitution of a fully functional naïve T-cell pool in the patient thymus.However,it is challenging to produce HTLPs in the high numbers required to meet clinical needs.Here,we found that adding tumor necrosis factor alpha(TNFα)to a DL-4-based culture system led to the generation of a large number of nonmodified or genetically modified HTLPs possessing highly efficient in vitro and in vivo T-cell potential from either CB HSPCs or mPB HSPCs through accelerated T-cell differentiation and enhanced HTLP cell cycling and survival.This study provides a clinically suitable cell culture platform to generate high numbers of clinically potent nonmodified or genetically modified HTLPs for accelerating immune recovery after HSCT and for T-cell-based immunotherapy(including CAR T-cell therapy).

T cell regeneration: an update on progress and challenges

T cells play essential roles in antitumor therapy.Via gene engineering technique to enhance tumor-antigen specificity,patient peripheral blood-derived T cells(PBT)show encouraging clinical outcomes in treating certain blood malignancies.However,the high costs,functionality exhaustion,and disease-condition-dependent availability of PBT prompt the attempts of exploring alternative T cell sources.Theoretically,induced T cells from pluripotent stem cells(PSC)are ideal candidates that integrate plenty of advantages that primary T cells lack,including unlimited off-the-shelf cell source and precision gene editing feasibility.However,researchers are still struggling with developing a straightforward protocol to induce functional and immunocompetent human T cells from PSC.Based on stromal cell-expressing or biomaterial-presenting Notch ligands DLL1 or DLL4,natural and induced blood progenitors can differentiate further toward T lineage commitment.However,none of the reported T induction protocols has yet translated into any clinical application,signaling the existence of numerous technical barriers for regenerating T cells functionally matching their natural PBT counterparts.Alternatively,new approaches have been developed to repopulate induced T lymphopoiesis via in vivo reprogramming or transplanting induced T cell precursors.Here,we review the most recent progress in the T cell regeneration field,and the remaining challenges dragging their clinical applications.