HUMAN PRIMITIVE HEMATOPOIETIC PROGENITOR CELLS ARE MORE ENRICHED IN CD34^+CD38^- POPULATION THAN IN CD34^+CD38^+ POPULATION

To clarify the hematopoietic potential of various sub-classes of human hematopoietic progenitor cells, we used a multicolor staining protocol in conjunction with anti-CD34 and -CD38 McAb. We characterized two cell fractions in CD34+cells with or without CD38 expression. A clonogenic assay showed that most CFC were present in CD34+CD38+ population. Morphologic analysis showed that blast-like cells were more enriched in the CD34+CD38 fraction. To clarify the biologic differences between both fractions, we examined the more primitive progenitor cell function by assessing long-term culture-initiating cells (LTC-IC) on the stromal cells. At the first two weeks, more CF.C harvested from the culture in the fractions initiated with both populations. However, more LTC-IC were present during weeks 4 to 12 in the CD34+CD38- population. These results indicate the primitive progenitors are more enriched in CD34+CD38 population than in CD34+CD38+ cells.

Role of osteoclasts in regulating hematopoietic stem and progenitor cells

Bone marrow(BM) cavities are utilized for hematopoiesis and to maintain hematopoietic stem cells(HSCs). HSCs have the ability to self-renew as well as to differentiate into multiple different hematopoietic lineage cells. HSCs produce their daughter cells throughout the lifespan of individuals and thus, maintaining HSCs is crucial for individual life. BM cavities provide a specialized microenvironment termed "niche" to support HSCs. Niches are composed of various types of cells such as osteoblasts, endothelial cells and reticular cells. Osteoclasts are unique cells which resorb bones and are required for BM cavity formation. Loss of osteoclast function or differentiation results in inhibition of BM cavity formation, an osteopetrotic phenotype. Osteoclasts are also reportedly required for hematopoietic stem and progenitor cell(HSPC) mobilization to the periphery from BM cavities. Thus, lack of osteoclasts likely results in inhibition of HSC maintenance and HSPC mobilization. However, we found that osteoclasts are dispensable for hematopoietic stem cell maintenance and mobilization by using three independent osteoclast-less animal models. In this review, I will discuss the roles of osteoclasts in hematopoietic stem cell maintenance and mobilization.

Involvement of VLA-5 and VLA-6 in facilitating endothelium-oriented transmigration of hematopoietic stem/progenitor cells

目的 :研究 VLA- 5及 VLA- 6是否参与内皮细胞促进的造血干 /祖细胞定向移行。方法 :对纯化人 CD34+ 细胞进行体外移行及阻断实验 ,观察其穿移覆盖人脐静脉内皮细胞 ( HUVECs)滤膜的能力。应用四色荧光活化流式细胞术 ( FACS)检测 CD34+细胞其粘附分子及趋化因子受体CXCR- 4的表达谱。结果 :基质由来因子 ( SDF) - 1 α介导的动员外周血 ( m PB)及骨髓 ( BM)来源的CD34+ 细胞穿透覆盖 HUVECs滤膜百分率分别为 ( 5 6.6± 2 0 .1 ) %及 ( 1 5 .6± 1 .8) % ,显著高于其穿移未覆盖 HUVECs滤膜的比率。预先对 CD34+ 细胞进行抗 VLA- 5和 /或 VLA- 6中和抗体处理可消除这一促进效应。此外 ,BM来源的 CD34+细胞其穿移覆盖及未覆盖 HUVECs滤膜的能力均显著低于 m PB CD34+细胞 ,两者间穿移能力的差异与其 VLA- 5及 VLA- 6(而非 VLA- 4及趋化因子受体 CXCR- 4)抗原表达水平相关。结论 :VLA- 5和 VLA- 6参与 HUVECs促进 HS/PCs穿移能力。

Impact of T cells on hematopoietic stem and progenitor cell function:Good guys or bad guys?

When hematopoietic stem and progenitor cells(HSPC)are harvested for transplantation, either from the bone marrow or from mobilized blood, the graft contains a significant number of T cells. It is these T cells that are the major drivers of graft-vs-host disease(Gv HD). The risk for Gv HD can simply be reduced by the removal of these T cells from the graft. However, this is not always desirable, as this procedure also decreases the engraftment of the transplanted HSPCs and, if applicable, a graft-vs-tumor effect. This poses an important conundrum in the field: T cells act as a double-edged sword upon allogeneic HSPC transplantation, as they support engraftment of HSPCs and provide anti-tumor activity, but can also cause Gv HD. It has recently been suggested that T cells also enhance the engraftment of autologous HSPCs, thus supporting the notion that T cells and HSPCs have an important functional interaction that is highly beneficial, in particular during transplantation. The underlying reason on why and how T cells contribute to HSPC engraftment is still poorly understood. Therefore, we evaluate in this review the studies that have examined the role of T cells during HSPC transplantation and the possible mechanisms involved in their supporting function. Understanding the underlying cellular and molecular mechanisms can provide new insight into improving HSPC engraftment and thus lower the number of HSPCs required during transplantation. Moreover, it could provide new avenues to limit the development of severe Gv HD, thus making HSPC transplantations more efficient and ultimately safer.

Hematopoietic stem cells in research and clinical applications:The“CD34 issue”

In this paper,experimental findings concerning the kinetics of hematopoietic reconstitution are compared to corresponding clinical data.Although not clearly apparent,the transplantation practice seems to confirm the basic proposals of experimental hematology concerning hematopoietic reconstitution resulting from successive waves of repopulation stemming from different subpopulations of progenitor and stem cells.One of the "f irst rate" parameters in clinical transplantations in hematology;i.e.the CD34+ positive cell dose,has been discussed with respect to the functional heterogeneity and variability of cell populations endowed by expression of CD34.This parameter is useful only if the relative proportion of stem and progenitor cells in the CD34+ cell population is more or less maintained in a series of patients or donors.This proportion could vary with respect to the source,pathology,treatment,processing procedure,the graft ex vivo treatment and so on.Therefore,a universal dose of CD34+ cells cannot be def ined.In addition,to avoid further confusion,the CD34+ cells should not be named "stem cells" or "progenitor cells" since these denominations only concern functionally characterized cell entities.

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.

To stay or to leave: Stem cells and progenitor cells navigating the S1P gradient

Most hematopoietic stem progenitor cells (HSPCs) reside in bone marrow (BM), but a small amount of HSPCs have been found to circulate between BM and tissues through blood and lymph. Several lines of evidence suggest that sphingosine-1-phosphate (S1P) gradient triggers HSPC egression to blood circulation after mobilization from BM stem cell niches. Stem cells also visit certain tissues. After a temporary 36 h short stay in local tissues, HSPCs go to lymph in response to S1P gradient between lymph and tissue and eventually enter the blood circulation. S1P also has a role in the guidance of the primitive HSPCs homing to BM in vivo, as S1P analogue FTY720 treatment can improve HSPC BM homing and engraftment. In stress conditions, various stem cells or progenitor cells can be attracted to local injured tissues and participate in local tissue cell differentiation and tissue rebuilding through modulation the expression level of S1P1, S1P2 or S1P3 receptors. Hence, S1P is important for stem cells circulation in blood system to accomplish its role in body surveillance and injury recovery.

Regulatory role of sphingosine kinase and sphingosine-1-phosphate receptor signaling in progenitor/stem cells

Balanced sphingolipid signaling is important for the maintenance of homeostasis. Sphingolipids were demonstrated to function as structural components, second messengers, and regulators of cell growth and survival in normal and disease-affected tissues. Particularly, sphingosine kinase 1 (SphK1) and its product sphingosine-1-phosphate (S1P) operate as mediators and facilitators of proliferation-linked signaling. Unlimited proliferation (selfrenewal) within the regulated environment is a hallmark of progenitor/stem cells that was recently associated with the S1P signaling network in vasculature, nervous,muscular, and immune systems. S1P was shown to regulate progenitor-related characteristics in normal and cancerstemcells(CSCs) viaG-protein coupled receptorsS1Pn(n=1 to 5). The SphK/S1P axis is crucially involved in the regulation of embryonic development of vasculature and the nervous system, hematopoietic stem cell migration, regeneration of skeletal muscle, and development of multiple sclerosis. The ratio of the S1P receptor expression, localization, and specific S1P receptoractivated downstream effectors influenced the rate of selfrenewal and should be further explored as regeneration related targets. Considering malignant transformation,it is essential to control the level of self-renewal capacity.Proliferation of the progenitor cell should be synchronized with differentiation to provide healthy lifelong function of blood, immune systems, and replacement of damaged ordead cells. The differentiation-related role of SphK/S1P remains poorly assessed. A few pioneering investigations exploredpharmacologicaltoolsthattargetsphingolipid signaling and can potentially confine and direct self-renewal towards normal differentiation. Further investigation is required to test the role of the SphK/S1P axis in regulation of self-renewal and differentiation.

The combination of epidermal growth factor and glycogen synthase kinase 3 inhibitor support long-term self-renewal of Sca-1 positive hepatic progenitor cells from normal adult mice

Isolation and long-term maintenance of hepatic progenitor cells (HPCs) from healthy, non-injured adult livers remains challenging due to the lack of specific surface markers for selection and a limited understanding of the mechanisms for maintaining self-renewal. Previously, we identified a Sca-1 positive, bipotent HPC population in the peri-portal region of adult liver, and found MAPK/ERK and Wnt/β-Catenin pathways to be synergistically involved in their proliferation. In this study, we report the long-term culture of Sca-1 positive HPCs with epidermal growth factor (EGF) and CHIR99021, a small molecule inhibitor of glycogen synthase kinase 3 (GSK-3). Sca-1+ HPCs remain non-tumorigenic when passaged 35 times in vitro over 1 year. Flow cytometric analysis indicates that HPCs are positive for Sca-1 and putative liver progenitor cell markers, including CD13, CD24 and Prominin-1, but negative for hematopoietic/endothelial cell markers CD31, CD34, CD45, CD90 and CD117. Immunocyto-chemistry and RT-PCR indicate Sca-1+ HPCs express albumin (ALB), α-fetoprotein (AFP), cytokeratin19 (CK19), Sox9 and a panel of special hepatic progenitor transcriptional factors. Moreover, Sca-1+ HPCs are able to differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions in vitro and can take part in liver repopulation in an acetaminophen (APAP) induced liver injury mouse model. This study provides a paradigm to capture and maintain HPCs from naive liver tissue and offers a valuable cell model for investigating the molecular mechanisms underlying the cell lineage relationship in normal liver.

Darbepoietin-Alfa after High-Dose Melphalan and Autologous Hemopoietic Progenitor Cell in Multiple Myeloma Patients: A Pilot Study

We describe the effects of Darbepoietin-alfa (Darbe) administration in Multiple Myeloma (MM) after autologous he-mopoietic progenitor cell transplantation (AHPCT). 26 MM patients undergoing AHPCT entered this study. 34 hemo-globin (Hb)-matched patients who had not received recombinant human erythropoietin (Epo) or Darbe and were treated with the same protocol were retrospectively selected for comparative data. Darbe (150 micro g total dose/weekly) was initiating in four weeks after AHPCT, with the aim of achieving an Hb level of ≥11 g/dl. The time to response to Darbe therapy was longer in the patients with Hb < 10 g/dL (p = 0.05) and with endogenous Epo levels ≤ 50 mU/ml (p = 0.0098). Hb level on day 60 and 90 after AHPCT, was faster for Darbe recipients (12.5, range 9.4 - 15.4, vs 10.6, range 8.8 - 13.4 g/dL, p = 0.0001, and 13.5, range 12.3 - 14.3, vs 12, range 9.8 - 14 g/dL, respectively, p = 0.0001). The need for Red Blood Cells transfusion, included in the period of 30 - 90 days post- AHPCT was similar (p = ns). This study demonstrates the accelerating effect of Darbe on Hb increase in the setting evaluated and shows that this effect signify- cantly depends on the endogenous Epo level at the start of treatment. The strategy of giving Darbe around 1 month after high-dose melphalan (HDM) doesn’t reduce RBC transfusion requirement.

Divergent expression of Neurl3 from hemogenic endothelial cells to hematopoietic stem progenitor cells during development

Prior to the generation of hematopoietic stem cells(HSCs)from the hemogenic endothelial cells(HECs)mainly in the dorsal aorta in midgestational mouse embryos,multiple hematopoietic progenitors including erythro-myeloid progenitors and lymphoid progenitors are generated from yolk sac HECs.These HSCindependent hematopoietic progenitors have recently been identified as major contributors to functional blood cell production until birth.However,little is known about yolk sac HECs.Here,combining integrative analyses of multiple single-cell RNA-sequencing datasets and functional assays,we reveal that Neurl3-EGFP,in addition to marking the continuum throughout the ontogeny of HSCs from HECs,can also serve as a single enrichment marker for yolk sac HECs.Moreover,while yolk sac HECs have much weaker arterial characteristics than either arterial endothelial cells in the yolk sac or HECs within the embryo proper,the lymphoid potential of yolk sac HECs is largely confined to the arterial-biased subpopulation featured by the Unc5b expression.Interestingly,the B lymphoid potential of hematopoietic progenitors,but not for myeloid potentials,is exclusively detected in Neurl3-negative subpopulations in midgestational embryos.Taken together,these findings enhance our understanding of blood birth from yolk sac HECs and provide theoretical basis and candidate reporters for monitoring step-wise hematopoietic differentiation.

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.

嵌合抗原受体介导的抗肿瘤细胞治疗的发展

嵌合抗原受体(CAR)技术驱动的T细胞疗法在肿瘤治疗中展示了巨大的创新潜力,尤其在血液肿瘤方面取得了显著成果。通过精准改造患者或供体细胞,使其能够特异性识别并清除肿瘤细胞,此策略已进入临床实践的新阶段。尽管如此,CAR-T细胞疗法在实体瘤中的治疗效果尚未达到预期,并且其潜在的不良反应引起了广泛关注。随着科技的不断进步,基于CAR技术改造的多种细胞类型,如NK细胞、巨噬细胞、NKT细胞及γδT细胞等,正在被纳入肿瘤治疗研究,拓展了治疗前景。本综述深入探讨了CAR技术的最新进展及其在细胞疗法中的应用,为抗肿瘤治疗提供潜在的新思路和可能性。

脐带血造血干细胞体外生成红细胞过程中高效脱核体系的研究

目的 研究磷脂酰肌醇三羟激酶(PI3K)、组蛋白去乙酰化酶2(HDAC2)和细胞松弛素B对脐带血造血干细胞体外培养促红系脱核的作用,以期建立高效脱核体系。方法 采用磁分选富集脐带血CD34+细胞。在培养第0、4天添加干细胞因子(SCF)、IL-3、促红细胞生成素(EPO),培养第7天添加SCF、EPO,培养第11、15、18天仅添加EPO。分别于第7、11、15和18天添加PI3K(0、25、50、100 ng/mL)、HDAC2(0、60、150、300 ng/mL)、细胞松弛素B(0、25、50、75 ng/μL)3种脱核剂。采用正交设计实验分析3种脱核剂的浓度和添加时间4因素4水平对促红系脱核效果的影响,获得最佳脱核条件并作为优选组,以不加脱核剂培养作为对照组进行验证。细胞培养至第21天时,用流式细胞仪分别检测CD235+、SYTO-64-细胞,计算脱核率。使用血细胞计数仪检测RBC,ELISA法检测2,3-二磷酸甘油(2,3-DPG)、化学发光法检测ATP,观察细胞形态。结果 最佳脱核条件为培养第15天添加PI3K浓度为100 ng/mL、HDAC2浓度为150 ng/mL和松弛素B浓度为75 ng/μL。培养至第21天,优选组红细胞脱核率为(74.30±5.59)%,对照组为(28.30±14.10)%,优选组高于对照组(t=9.099,P=0.012)。培养体系获得RBC平均可达2×1010/L,红细胞ATP、2,3-DPG与正常红细胞无差异,红细胞形态与正常红细胞一致。结论 以上最佳脱核条件建立的培养体系可促进造血干细胞体外生成红细胞高效脱核。

黑枸杞花青素联合人脂肪源性血管外膜细胞支持脐血造血干/祖细胞的增殖

背景:黑枸杞花青素(Anthocyanins in Lycium ruthenicum Murr,ALRM)是黑枸杞中重要的活性成分之一,具有抗氧化、免疫调节等功效。人脂肪源性血管外膜细胞(CD146^(+)hAD-PCs)是骨髓间充质干细胞的前体细胞,在体外具有促进造血干/祖细胞增殖与分化的功能。ALRM联合CD146^(+)hAD-PCs对脐血造血干/组细胞的体外支持作用有待于研究。目的:探讨ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞体外扩增的支持作用。方法:CCK-8法检测不同质量浓度ALRM(0,200,400,600,800,1000 mg/L)对CD146^(+)hAD-PCs增殖的影响;流式细胞术检测ALRM对CD146^(+)hAD-PCs细胞周期的影响。共培养实验分为空白组、ALRM组、CD146^(+)hAD-PCs组、ALRM+CD146^(+)hAD-PCs组,分析ALRM联合CD146^(+)hAD-PCs对脐血CD34^(+)造血干/祖细胞的体外支持作用。共培养1,2,4周,比较扩增后细胞数量、集落形成单位数量,流式细胞仪检测细胞免疫表型,ELISA检测细胞因子水平。结果与结论:(1)ALRM质量浓度为200 mg/L时,CD146^(+)hAD-PCs活力最高,CD146^(+)hAD-PCs的G_(0)/G_(1)期细胞比例下降,S期、G_(2)/M期细胞比例上升(P<0.01)。(2)脐血CD34^(+)造血干/祖细胞数量变化:在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组高于ALRM组(P均<0.05),在共培养2,4周时ALRM+CD146^(+)hAD-PCs组高于CD146^(+)hAD-PCs组(P均<0.05),ALRM组与空白组随着共培养时间延长细胞数量逐渐减少。(3)集落形成能力及免疫表型分析:在共培养1,2周时ALRM+CD146^(+)hAD-PCs组的集落形成单位数量高于CD146^(+)hAD-PCs组和ALRM组(P均<0.05);在共培养1,2,4周时ALRM+CD146^(+)hAD-PCs组CD45^(+)、CD34^(+)CD33^(-)细胞比例高于CD146^(+)hAD-PCs组(P均<0.01)。(4)细胞因子变化:在共培养4周时ALRM+CD146^(+)hAD-PCs组的白细胞介素2水平高于ALRM组、CD146^(+)hAD-PCs组(P<0.05);在共培养2,4周时ALRM+CD146^(+)hAD-PCs组白细胞介素3水平高于CD146^(+)hAD-PCs组(P<0.05);在共培养1周时ALRM+CD146^(+)h AD-PCs组的粒细胞集落刺激因子水平高于CD146^(+)hAD-PCs组,在共培养2周时高于ALRM组、CD146^(+)hAD-PCs组(P<0.01);在共培养1,2,4周时ALRM组、ALRM+CD146^(+)hAD-PCs组的干扰素γ水平低于CD146^(+)hAD-PCs组(P<0.05)。(5)由于空白组无基质细胞,脐血CD34^(+)造血干/祖细胞在共培养1周之后就无法计数,未进行免疫表型、集落分析和细胞因子检测。(6)结果表明:ALRM可以通过促进CD146^(+)hAD-PCs增殖和细胞周期转化进而促进脐血CD34^(+)造血干/祖细胞的体外扩增,在造血干细胞移植研究方面具有重要价值。

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.

Bone marrow mononuclear cells for joint therapy: The role of macrophages in inflammation resolution and tissue repair

Osteoarthritis(OA)is the most prevalent joint disease causing major disability and medical expenditures.Synovitis is a central feature of OA and is primarily driven by macrophages.Synovial macrophages not only drive inflammation but also its resolution,through a coordinated,simultaneous expression of pro-and anti-inflammatory mechanisms that are essential to counteract damage and recover homeostasis.Current OA therapies are largely based on anti-inflammatory principles and therefore block pro-inflammatory mechanisms such as prostaglandin E2 and Nuclear factor-kappa B signaling pathways.However,such mechanisms are also innately required for mounting a pro-resolving response,and their blockage often results in chronic low-grade inflammation.Following minor injury,macrophages shield the damaged area and drive tissue repair.If the damage is more extensive,macrophages incite inflammation recruiting more macrophages from the bone marrow to maximize tissue repair and ultimately resolve inflammation.However,sustained damage and inflammation often overwhelms pro-resolving mechanisms of synovial macrophages leading to the chronic inflammation and related tissue degeneration observed in OA.Recently,experimental and clinical studies have shown that joint injection with autologous bone marrow mononuclear cells replenishes inflamed joints with macrophage and hematopoietic progenitors,enhancing mechanisms of inflammation resolution,providing remarkable and long-lasting effects.Besides creating an ideal environment for resolution with high concentrations of interleukin-10 and anabolic growth factors,macrophage progenitors also have a direct role in tissue repair.Macrophages constitute a large part of the early granulation tissue,and further transdifferentiate from myeloid into a mesenchymal phenotype.These cells,characterized as fibrocytes,are essential for repairing osteochondral defects.Ongoing“omics”studies focused on identifying key drivers of macrophagemediated resolution of joint inflammation and those required for efficient osteochondral repair,have the potential to uncover ways for developing engineered macrophages or off-the-shelf pro-resolving therapies that can benefit patients suffering from many types of arthropaties,not only OA.

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.