Development and analysis of transgenic mice expressing porcine hematopoietic cytokines: a model for achieving durable porcine hematopoietic chimerism across an extensive xenogeneic barrier

Abstract: The capacity of mixed hematopoietic chimerism to induce tolerance has not been demonstrated in discordant xenogeneic species combinations because of the difficulty in achieving lasting hematopoietic engraftment. In an effort to create a model of long‐lasting disparate xenogeneic hematopoietic chimerism, we have developed transgenic (Tg) mice carrying porcine cytokines. Three lines of Tg mice were generated: one carrying porcine IL‐3 and GM‐CSF genes only (termed IL/GM) and the remaining two lines carrying in addition, the soluble SCF gene (termed IL/GM/sS) or membrane‐bound SCF gene (termed IL/GM/mS). Sera from mice with IL/GM and IL/GM/sS transgenes markedly stimulated the proliferation of swine marrow cells in vitro. However, proliferation of swine marrow cells was not induced in cultures containing IL/GM/mS sera. Consistent with these observations, ELISA assays revealed detectable levels of porcine cytokines in the sera of IL/GM and IL/GM/sS, but not in sera of IL/GM/mS Tg mice. Marrow stromal cells prepared from all three kinds of Tg mice, but not those from non‐Tg littermates, were capable of supporting the growth of porcine hematopoietic cells in vitro. Immunodeficient Tg mice were generated by crossing Tg founders with C.B‐17 SCID mice for five generations. All Tg immunodeficient mice showed improved porcine hematopoietic engraftment compared with non‐Tg controls. These Tg mice provide a useful model system for studying porcine hematopoietic stem cells, and for evaluating the feasibility of donor‐specific tolerance induction by mixed chimerism across highly disparate xenogeneic barriers.     

Xenogeneic transplantation of human WJ‐MSCs rescues mice from acute radiation syndrome via Nrf‐2‐dependent regeneration of damaged tissues

There is an unmet medical need for radiation countermeasures that can be deployed for treatment of exposed individuals during ionizing radiation (IR) accidents or terrorism. Wharton's jelly mesenchymal stem cells (WJ‐MSCs) from human umbilical cord have been shown to avoid allorecognition and induce a tissue‐regenerating microenvironment, which makes them an attractive candidate for mitigating IR injury. We found that WJ‐MSCs protected mice from a lethal dose of IR even when transplanted up to 24 hours after irradiation, and a combination of WJ‐MSCs and antibiotic (tetracycline) could further expand the window of protection offered by WJ‐MSCs. This combinatorial approach mitigated IR‐induced damage to the hematopoietic and gastrointestinal system. WJ‐MSCs increased the serum concentration of the cytoprotective cytokines granulocyte colony‐stimulating factor (G‐CSF) and IL‐6 in mice. Knockdown of G‐CSF and IL‐6 in WJ‐MSCs before injection to lethally irradiated mice or transplantation of WJ‐MSCs to lethally irradiated Nrf‐2 knockout mice significantly nullified the therapeutic protective efficacy. Hence, WJ‐MSCs could be a potential cell‐based therapy for individuals accidentally exposed to radiation.     

A novel model of pneumonia from intraperitoneal injection of bacteria

BACKGROUND: Pneumonia remains a major clinical problem in the surgical patient. Experimental modeling by intratracheal injection of bacteria is not consistently reproducible. In an attempt to produce peritonitis by Klebsiella, we found evidence of pneumonia on autopsy and further developed this approach as a new experimental model. METHODS: Male Swiss Webster mice were given intraperitoneal (IP) injections of Klebsiella pneumoniae serotype 2 in different doses and this was compared with similar doses given intravenously (IV). A dose dependent survival curve was generated. Subsequently, 10(3) colony forming units (CFU) of bacteria were used in further experiments. Blood, peritoneal fluid and lung tissue were collected at time points up to 72 hours after injection and were cultured for levels of bacteria. Lung weights and myeloperoxidase levels were also measured. RESULTS: Intraperitoneal administration of Klebsiella was uniformly lethal with as few as 10(2) bacteria. Lung weight increased after IP Klebsiella, and all animals became bacteremic within 24 hours correlating with high bacterial levels in the lung. Conversely, most animals (72%) survived IV injection of bacteria, and were able to clear bacteria from the blood and lung. CONCLUSIONS: We found that this model produced no clinically apparent peritonitis after 48 hours, but uniformly resulted in histopathologic changes of pneumonia by 24 hours. Survival time was related to initial dose of Klebsiella and there was a linear correlation between bacterial levels in the blood and lung. This model is reproducible, simple to perform, and the severity is easy to manipulate.     

人脐血造血干/祖细胞的生物反应器大规模扩增及移植实验

目的 利用生物反应器大规模扩增人脐血造血干/祖细胞,并通过动物移植实验检验该方法的有效性.方法 采集抗凝脐血10份,分离出单个核细胞(MNC),分别进行生物反应器扩增培养和静态扩增培养.检测扩增前后细胞表面CD34、CD38、CD133、CD184和CD62L分子的表达,并进行造血干/祖细胞集落的培养.取非肥胖糖尿病重症联合免疫缺陷小鼠,以X射线照射后,分为4组,其中MNC组小鼠注射未经扩增培养的MNC;静态扩增组小鼠注射经过静态扩增培养的细胞;反应器扩增组小鼠注射经过生物反应器扩增培养的细胞;空白对照组小鼠注射生理盐水.移植后6周处死存活小鼠,收集骨髓细胞,检测其中CD45+、CD3+、CD19+和CD33+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 生物反应器扩增前MNC为(1.2～2.8)×108个,扩增后为(3.7～12.6)×108个,扩增后的细胞数明显高于静态扩增培养者(P<0.01).经生物反应器扩增后所形成的红系集落形成单位、粒-巨噬细胞集落形成单位数明显高于经静态扩增者(P<0.05).移植6周后,空白对照组小鼠均死亡,MNC组存活率为35%,静态扩增组存活率为30%,反应器扩增组存活率为62.9%,后者明显高于前二者(P<0.05).各组存活小鼠骨髓细胞中均检测到Alu基因和Cart-Ⅰ基因的表达以及人源CD33+、CD45+、CD3+及CD19+细胞.结论 利用生物反应器可大规模扩增人脐血造血干/祖细胞,所得细胞能植入小鼠体内,并能获得造血功能重建.     

Optimising proliferation and migration of mesenchymal stem cells using platelet products: A rational approach to bone regeneration

This study investigates how mesenchymal stem cell's (MSCs) proliferation and migration abilities are influenced by various platelet products (PP). Donor‐matched, clinical‐, and control laboratory‐standard PPs were generated and assessed based on their platelet and leukocyte concentrations. Bone marrow derived MSCs were exposed to these PP to quantify their effect on in vitro MSC proliferation and migration. An adapted colony forming unit fibroblast (CFU‐F) assay was carried out on bone marrow aspirate using clinical‐standard PP‐loaded electrospun poly(?‐caprolactone) (PCL) membrane to mimic future clinical applications to contain bone defects. Clinical‐standard PP had lower platelet (2.5 fold, p < 0.0001) and higher leukocyte (14.1 fold, p < 0.0001) concentrations compared to laboratory‐standard PP. It induced suboptimal MSC proliferation compared to laboratory‐standard PP and fetal calf serum (FCS). All PP induced significantly more MSC migration than FCS up to 24 h. The removal of leukocytes from PP had no effect on MSC proliferation or migration. The PP‐loaded membranes successfully supported MSC colony formation. This study indicates that platelet concentrations in PP impact MSC proliferation more than the presence of leukocytes, whilst MSC migration in response to PP is not influenced by platelet or leukocyte numbers. Clinical‐standard PP could be applied alongside manufactured membranes in the future treatment of bone reconstruction. © 2019 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:1329–1338, 2019.     

Increased marrow-derived osteoprogenitor cells and endosteal bone formation in mice lacking thrombospondin 2.

The phenotype of thrombospondin 2 (TSP2)-null mice includes abnormalities in collagen fibrils and increases in ligamentous laxity, vascular density, and bleeding time. In this study, analyses by computerized tomography (CT) revealed that cortical density was increased in long bones of TSP2-null mice. Histomorphometric analysis showed that the mid-diaphyseal endosteal bone formation rate (BFR) of TSP2-null mice was increased in comparison with that of wild-type (WT) animals. Although microgeometric analysis showed that periosteal and endosteal radii were reduced, the mechanical properties of femurs from TSP2-null mice were not significantly different from those of controls, presumably because of the concomitant increase in endosteal bone mass. Bone loss in ovariectomized mice was equivalent for WT and mutant mice, a finding that indicates that TSP2-null animals are capable of normal bone resorption. To further explore the cellular basis for the increased endosteal BFR in TSP2-null mice, marrow stromal cells (MSCs) were isolated and examined in vitro. These cells were found to be present in increased numbers in a colony forming unit (CFU) assay and showed an increased rate of proliferation in vitro. We conclude that TSP2 regulates the proliferation of osteoblast progenitors, directly or indirectly, and that in its absence endosteal bone formation is increased.     

低剂量粒细胞巨噬细胞集落刺激因子诱导的小鼠骨髓未成熟树突状细胞抗成熟特性的研究

目的观察内毒素／脂多糖(LPS)、肿瘤坏死因子α(TNF-α)、干扰素1(IFN-γ)等促成熟物质对低剂量粒细胞巨噬细胞集落刺激因子(rmGM-CSF)诱导的小鼠骨髓未成熟树突状细胞(DC)成熟特性的影响。方法制备小鼠骨髓细胞，分别用不同剂量rmGM-CSF培养，6d后收集悬浮细胞进行检测。用LPS、TNF-α、IFN-1与小剂量rmGM-CSF培养获得的Dc(GM^low DC)共同孵育3d后，行混合淋巴细胞反应，观察其诱导未致敏脾淋巴细胞增殖的情况，并与大剂量rmGM-CSF培养获得的Dc(GM^high DC)进行比较。结果GM^lew DC不能激活未致敏脾淋巴细胞，且在与LPS、TNF-α和IFN-1共同培养3d后，仍不能有效诱导未致敏脾淋巴细胞增殖，刺激指数(SI)均＜2．00；而GM^high DC刺激未致敏脾淋巴细胞增殖的能力较强，SI为4．71。结论GM^lew DC具有对LPS、TNF-α和IFN-1刺激不敏感的抗成熟特性。     

混合脐血移植后造血及免疫重建特性的研究

目的　探讨混合脐血移植重建造血和免疫功能的特点及规律。方法　分别将两份人HLA半相合、不相合混合脐血或单份脐血输入经亚致死量照射后的严重联合免疫缺陷 (SCID)小鼠。观察三组脐血在SCID小鼠体内植入状况及造血和免疫功能重建特性。结果　单份和混合脐血均可在受鼠体内植入 ,形成供 受混合嵌合体 ,植入率差异无显著性 (P >0 .0 5 ) ,并能重建人类多系造血 ,且易于向NK和B淋巴细胞方向分化。用多聚酶链反应 序列特异性寡核苷酸 (PCR SSO)探针检测人HLA DQB1基因发现 ,混合脐血移植可有 1份或 2份脐血植入 ,其中造血祖细胞含量和体外集落形成能力高者 ,更易于植入 ,且供者间HLA差异小者 ,趋向两份脐血同时植入。结论　混合脐血移植可重建SCID小鼠造血和免疫功能 ,但造血细胞各谱系分化不均衡。     

Induction of chimerism in mice using human MHC class I-mismatched Hoechst 33342 side population donor stem cells

A population of Hoechst 33342-stained cells, termed side population (SP) cells, can reconstitute the hematopoietic system of syngeneic mice. This study examined whether limiting numbers of SP cells can repopulate mice across a xenogeneic MHC class I barrier. SP cells were isolated from HLA.B7 and HLA.A2.1 transgenic mice by FACS and placed in colony assays or transplanted into irradiated C57BL/6 (B/6) recipients. SP cells contained few colony-forming cells when placed directly in culture. The number of GM-CFC and HPP-CFC increased up to 3000- and 300-fold, respectively, after 7 days in IL-3- and SCF-stimulated liquid culture. BMC-derived GM-CFC increased up to only 12-fold and HPP-CFC decreased after 7 days in culture. HLA-B7 SP cells (2500-5000) were transplanted into lethal-irradiated B/6 mice. Two-color flow analysis, 4-6 weeks after transplantation, showed that HLA-B7 expression in granulocyte-, macrophage-, and lymphocyte-specific lineages from reconstituted mice was similar to that in B7 transgenic mice. Secondary transplanted B/6 mice also showed a pattern of HLA-B7 expression similar to that in transgenic mice and were followed for longer than 16 weeks with stable chimerism. When HLA-A2.1 SP cells were transplanted into sublethally irradiated mice, 50% of the mice expressed HLA-A2 by PCR analysis in short-term repopulation studies. These data confirm that limiting numbers of SP cells can repopulate the major hematopoietic lineages in lethal and sublethally irradiated mice across a human MHC class I barrier. Therefore, SP cells may be useful for establishing mixed chimerism, which may induce immunologic nonresponsiveness to donor antigens in solid organ transplantation.     

Improvement of the survival rate by fetal liver cell transplantation in a mice lethal liver failure model

BACKGROUND: The use of cell transplantation as an alternative therapy for orthotopic liver transplantation has been widely anticipated due to a chronic donor shortage. We previously reported the method used to enrich hepatic progenitor cells (HPCs) forming cell aggregations. In this study, we transplanted HPCs into the liver injury model mice to determine whether HPC transplantation may improve the liver dysfunction. METHODS: We obtained donor cells from E13.5 fetal livers of green fluorescent protein (GFP) transgenic mice. We transplanted GFP-positive fetal liver cells into the transgenic mice which express diphtheria toxin (DT) receptors under the control of an albumin enhancer/promoter. Subsequently, we induced selective liver injury to recipient mice by DT administration. We then evaluated the engraftment of the transplanted cells and their effect on survivorship. RESULTS: The low dose of DT induced sublethal liver injury and the high dose of DT was lethal to the liver injury model mice. The transplanted GFP-positive cells were engrafted into the recipient livers and expressed albumin, resembling mature hepatocytes. They continued to proliferate, forming clusters. The survival rate at 25 days after transplantation of the cell-transplanted group (8 of 20; 40.0%) was improved significantly (P=0.0047) in comparison to that of the sham-operated group (0 of 20; 0%). CONCLUSIONS: The transplanted cells were engrafted and repopulated the liver of recipient mice, resulting in the improvement of the survival rate of the liver injury model mice. We therefore propose that HPCs are a desirable cell source for cell transplantation.     

Glycogen synthase kinase‐3α/β inhibition promotes in vivo amplification of endogenous mesenchymal progenitors with osteogenic and adipogenic potential and their differentiation to the osteogenic lineage

Small molecules are attractive therapeutics to amplify and direct differentiation of stem cells. They also can be used to understand the regulation of their fate by interfering with specific signaling pathways. Mesenchymal stem cells (MSCs) have the potential to proliferate and differentiate into several cell types, including osteoblasts. Activation of canonical Wnt signaling by inhibition of glycogen synthase kinase 3 (GSK‐3) has been shown to enhance bone mass, possibly by involving a number of mechanisms ranging from amplification of the mesenchymal stem cell pool to the commitment and differentiation of osteoblasts. Here we have used a highly specific novel inhibitor of GSK‐3, AR28, capable of inducing β‐catenin nuclear translocation and enhanced bone mass after 14 days of treatment in BALB/c mice. We have shown a temporally regulated increase in the number of colony‐forming units–osteoblast (CFU‐O) and –adipocyte (CFU‐A) but not colony‐forming units–fibroblast (CFU‐F) in mice treated for 3 days. However, the number of CFU‐O and CFU‐A returned to normal levels after 14 days of treatment, and the number of CFU‐F was decreased significantly. In contrast, the number of osteoblasts increased significantly only after 14 days of treatment, and this was seen together with a significant decrease in bone marrow adiposity. These data suggest that the increased bone mass is the result of an early temporal wave of amplification of a subpopulation of MSCs with both osteogenic and adipogenic potential, which is driven to osteoblast differentiation at the expense of adipogenesis. © 2011 American Society for Bone and Mineral Research.     

Impact of maturational status on the ability of osteoblasts to enhance the hematopoietic function of stem and progenitor cells

Osteoblasts (OBs) exert a prominent regulatory effect on hematopoietic stem cells (HSCs). We evaluated the difference in hematopoietic expansion and function in response to co‐culture with OBs at various stages of development. Murine calvarial OBs were seeded directly (fresh) or cultured for 1, 2, or 3 weeks prior to seeding with 1000 Lin‐Sca1 + cKit+ (LSK) cells for 1 week. Significant increases in the following hematopoietic parameters were detected when comparing co‐cultures of fresh OBs to co‐cultures containing OBs cultured for 1, 2, or 3 weeks: total hematopoietic cell number (up to a 3.4‐fold increase), total colony forming unit (CFU) number in LSK progeny (up to an 18.1‐fold increase), and percentage of Lin‐Sca1+ cells (up to a 31.8‐fold increase). Importantly, these studies were corroborated by in vivo reconstitution studies in which LSK cells maintained in fresh OB co‐cultures supported a significantly higher level of chimerism than cells maintained in co‐cultures containing 3‐week OBs. Characterization of OBs cultured for 1, 2, or 3 weeks with real‐time PCR and functional mineralization assays showed that OB maturation increased with culture duration but was not affected by the presence of LSK cells in culture. Linear regression analyses of multiple parameters measured in these studies show that fresh, most likely more immature OBs better promote hematopoietic expansion and function than cultured, presumably more mature OBs and suggest that the hematopoiesis‐enhancing activity is mediated by cells present in fresh OB cultures de novo. © 2011 American Society for Bone and Mineral Research.     

Effects of ex vivo activated immune cells on syngeneic and semi-allogeneic bone marrow transplantation in mice

We have previously shown that a novel immunotherapy using ex vivo activated immune cells is capable of promoting survival and hematopoietic recovery in mice after combined chemotherapy and radiotherapy. In this study, we investigated whether the immunotherapy with ex vivo activated immune cells had the same beneficial effects after syngeneic and semiallogeneic bone marrow transplantation (BMT) in BALB/c mice subjected to a lethal dose of total body irradiation (TBI). Immune cells were cultured in vitro with a combination of cytokines and a calcium ionophore for 2 days and subsequently injected to mice daily for 4 days starting 1 day after BMT. The immunotherapy enhanced survival and multilineage peripheral blood recovery in BMT mice with limited numbers of transplanted bone marrow cells when a low dose of ex vivo activated immune cells were used. However, the beneficial effects were completely lost when a higher dose of the same therapeutic immune cells were tested, and instead the immunotherapy significantly exacerbated complications associated with the lethal radiation and BMT. This detrimental effect appeared to be the result of strong in vivo nonspecific immune responses induced by either activated therapeutic immune cells or interaction between therapeutic immune cells and MHC-mismatched bone marrow cells transplanted or both. Our data suggest that the immunotherapy with appropriately selected dosages may be beneficial to BMT but vigorous in vivo immune responses soon after BMT may exacerbate post-transplant complications.     

HSCT联合不同剂量内皮祖细胞输注对小鼠造血重建的影响

目的 探讨异基因造血干细胞移植联合不同剂量内皮祖细胞(EPC)输注对移植后造血重建的影响.方法 以C57BL/6小鼠为供鼠,Balb/c小鼠为受鼠,进行HSCT,输注骨髓单个核细胞数量为5×106个/只.仅进行HSCT者为单纯骨髓细胞移植组;行HSCT的同时经尾静脉输注供者骨髓单个核细胞诱导培养的EPC的受鼠为EPC联合移植组,EPC的输注量分别为5×104、1×105、5× 105和1×106个/只.另设正常对照组和致死量照射组.观察小鼠的存活率、造血重建情况及骨髓微环境的变化.结果 各EPC联合移植组小鼠存活时间长于单纯骨髓移植组,5×105 EPC联合移植组至观察结束时存活率为100％,高于其他各组(P＜0.05).移植后10和15 d,5×105 EPC联合移植组外周血白细胞数量高于其他组(P＜0.05).移植后15 d,5×105 EPC联合移植组外周血血小板数量高于其他组(P＜0.05).5×105 EPC联合移植组造血组织增生程度也好于其他组.5×105 EPC联合移植组骨髓内HSC比例为(1.06±0.03)％,高于其他各组(P＜0.05).结论 小鼠异基因骨髓移植中联合输注5×105 EPC能够有效促进造血重建,提高小鼠存活率.     

Effect of pig‐specific cytokines on mobilization of hematopoietic progenitor cells in pigs and on pig bone marrow engraftment in baboons

Kozlowski T, Monroy R, Giovino M, Hawley RJ, Glaser R, Li Z, Meshulam DH, Spitzer TR, Cooper DKC and Sachs DH Effect of pig specific cytokines on mobilization of hematopoietic progenitor cells in pigs and on pig bone marrow engraftment in baboons. Xenotransplantation 1999; 6: 00-00. ©Munksgaard, Copenhagen. Abstract: Mixed hematopoietic chimerism has been found to be a requirement for achieving specific immunologic hyporesponsiveness. Some of the requirements for in vitro and in vivo coexistence of discordant hematopoietic systems in the pig-to-baboon (or human) model have been investigated. We have tested the efficacy of pig-specific cytokines (PSC) (IL3, SCF, GM-CSF) in the mobilization of porcine bone marrow (BM) progenitors in vivo (i) in the pig and (ii) in baboons that underwent a conditioning regimen and porcine BM transplantation. In a preliminary in vitro study, porcine BM cells were incubated in various media to assess the effect of human plasma on pig progenitors in a colony-forming unit (CFU) assay. In in vivo studies, four pigs received PSC and one control pig did not. Six baboons underwent natural antibody removal, with subsequent pig BM transplantation. Four of these six underwent nonmyeloablative (n=2) or myeloablative (n=2) conditioning and all received PSC treatment. Two baboons did not receive PSC, one of which underwent a nonmyeloablative regimen. Sequential blood samples and BM biopsies in pigs and baboons were analyzed by CFU assay for the detection of porcine cells. Baboon samples were analyzed by polymerase chain reaction (PCR) to detect porcine DNA. In the case of the in vitro tests, colony forming by porcine progenitors was not inhibited by media containing human plasma and for the in vivo tests, PSC increased the number of progenitors in pig BM; mobilization of progenitors into the peripheral blood was observed. PSC-treated baboons which experienced transient depletion of leukocytes < 1,000/ml (as an effect of the conditioning regimen) had porcine BM cells detectable by PCR for as long as day 316 after BM transplantation. In conclusion we found that: (i) under the conditions of these studies, in vitro porcine progenitor cell growth was not inhibited by human plasma containing natural antibody and complement; (ii) PSC treatment led to an increased number of progenitors in pig BM and peripheral blood; (iii) the combination of an effective conditioning regimen and treatment with PSC was capable of inducing long-term survival of pig progenitors in baboons, although only a low level of engraftment was achieved.     

人脐血造血干/祖细胞的磁力搅拌悬浮培养及移植实验

目的 探讨磁搅拌大规模培养体系对人脐血造血祖细胞的扩增效果以及扩增的人造血祖细胞植入动物体内后的造血重建情况.方法 从新鲜抗凝脐血中分离出单个核细胞(MNC),以添加干细胞因子、酪氨酸激酶受体3配基及血小板生成素的无血清培养体系进行培养.静态扩增组的细胞置于T25培养瓶中培养,磁搅拌悬浮扩增组(磁搅拌扩增组)的细胞采用Celstir装置进行培养,培养体系为50～100 ml.培养7 d后进行细胞计数、集落培养检测和细胞表面分子表达的测定.以不进行培养者为对照组.非肥胖糖尿病重症联合免疫缺陷(NOD/SCID)小鼠在接受2.5 Gy的亚致死剂量X射线照射后分别从尾静脉输入上述静态扩增组、磁搅拌扩增组和对照组的MNC(5×106个),另设不移植的空白对照组.观察小鼠的存活情况,6周后处死存活小鼠,检测骨髓细胞中CD34+细胞、CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞的含量以及人特异的Cart-Ⅰ和Alu基因的表达.结果 经过7天的培养,磁搅拌扩增组的造血祖细胞扩增倍数为(2.8±0.45)倍,明显高于静态扩增组的(2.1±0.48)倍(P<0.01).磁搅拌扩增组形成的红系集落、粒-巨噬细胞集落数均明显高于静态扩增组(P<0.05).静态扩增组扩增后的CD34+细胞、CD34+CD38-细胞和CD133+细胞含量均高于磁搅拌扩增组(P<0.05),而CD184+细胞和CD62L+细胞含量低于磁搅拌扩增组(P<0.01).移植后6周,对照组、静态扩增组和磁搅拌扩增组分别有3、4、5只小鼠存活,三组间两两比较,6周存活率的差异无统计学意义(P>0.05).存活6周的小鼠,其骨髓中能检人特异性CD34+细胞,以及CD3+细胞、CD19+细胞、CD33+细胞及CD45+细胞,也检测到人Alu基因和Cart-Ⅰ基因的表达.结论 磁搅拌培养能大规模扩增脐带血造血祖细胞,扩增的细胞能植入x射线照射的NOD/SCID小鼠,并重建其多系造血.     

由脐血单个核细胞和富集的CD34+细胞扩增的造血干/祖细胞的生理功能比较

目的 探讨由脐血单个核细胞(MNC)和富集的CD34+细胞起始扩增所得的造血干/祖细胞在体内植入及造血重建的能力.方法 从人脐血中分离出MNC和CD34+细胞,在体外扩增7 d.将非肥胖糖尿病型重症联合免疫缺陷型(NOD/SCID)小鼠分为四组,在接受亚致死剂量铯源照射后,进行细胞移植,实验A组接受由MNC培养得到的CD34+细胞和CD34-细胞;实验B组接受由富集的CD34+细胞培养得到的CD34+细胞和CD34-细胞;阳性对照组接受从脐血新鲜分离的CD34+细胞和CD34-细胞;阴性对照组不接受细胞移植,仅输注相同体积的IMDM培养基.6周后处死存活的小鼠,取其骨髓、脾脏和外周血细胞,分别进行细胞表型分析、集落和人特异性基因的检测.结果 经过体外扩增,以富集的CD34+细胞起始培养者的细胞总扩增倍数为39.8倍,远高于以MNC为起始细胞者的1.88倍.移植6周后,所有接受细胞移植的小鼠均存活,存活小鼠的骨髓和脾脏细胞中均能检测到人源细胞(CD45+细胞)及人源的各系血细胞,实验A组各类细胞的含量稍高于实验B组,且接受细胞移植小鼠的骨髓和脾脏细胞中可检测出人特异的Alu序列.结论 与从脐血中新鲜分离的细胞相比,扩增后的造血干/祖细胞的体内植入能力有所下降,以MNC起始扩增的造血干/祖细胞体内植入能力优于以富集的CD34+细胞起始扩增者,但二者体内造血重建能力的差异不显著.     

Deletion of CD74, a putative MIF receptor,in mice enhances osteoclastogenesis and decreases bone mass

CD74 is a type II transmembrane protein that can act as a receptor for macrophage migration inhibitory factor (MIF) and plays a role in MIF‐regulated responses. We reported that MIF inhibited osteoclast formation and MIF knockout (KO) mice had decreased bone mass. We therefore examined if CD74 was involved in the ability of MIF to alter osteoclastogenesis in cultured bone marrow (BM) from wild‐type (WT) and CD74‐deficient (KO) male mice. We also measured the bone phenotype of CD74 KO male mice. Bone mass in the femur of 8‐week‐old mice was measured by micro–computed tomography and histomorphometry. Bone marrow cells from CD74 KO mice formed 15% more osteoclast‐like cells (OCLs) with macrophage colony‐stimulating factor (M‐CSF) and receptor activator of NF‐κB ligand (RANKL) (both at 30 ng/mL) compared to WT. Addition of MIF to WT cultures inhibited OCL formation by 16% but had no effect on CD74KO cultures. The number of colony forming unit granulocyte‐macrophage (CFU‐GM) in the bone marrow of CD74 KO mice was 26% greater than in WT controls. Trabecular bone volume (TBV) in the femurs of CD74 KO male mice was decreased by 26% compared to WT. In addition, cortical area and thickness were decreased by 14% and 11%, respectively. Histomorphometric analysis demonstrated that tartrate‐resistant acid phosphatase (TRAP)(+) osteoclast number and area were significantly increased in CD74 KO by 35% and 43%, respectively compared to WT. Finally, we examined the effect of MIF on RANKL‐induced‐signaling pathways in bone marrow macrophage (BMM) cultures. MIF treatment decreased RANKL‐induced nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and c‐Fos protein in BMM cultures by 70% and 41%, respectively. Our data demonstrate that CD74 is required for MIF to affect in vitro osteoclastogenesis. Further, the bone phenotype of CD74 KO mice is similar to that of MIF KO mice. MIF treatment of WT cultures suppressed RANKL‐induced activator protein 1 (AP‐1) expression, which resulted in decreased osteoclast differentiation in vitro. We propose that CD74 plays a critical role in the MIF inhibition of osteoclastogenesis. © 2013 American Society for Bone and Mineral Research.     

人脐血细胞体外扩增的实验研究和意义

目的：了解脐血造血干细胞在体外扩增培养过程中的生长分化特点,探讨脐血干细胞与体外扩增后的祖细胞混合移植的可行性以及合适的移植时机。方法：按种脐血单个核细胞（MNC）于含体积浓度为20％胎牛血清（FCS）的IMDM悬浮培养体系中,加入干细胞因子（SCF）、Flt-3配基（FL）、粒－单细胞集落刺激因子（GM－CSF）、白细胞介素－3（IL－3）、白细胞介素－6（IL－6） ,培养21d,每周半量换液,同时进行细胞计数,涂片形态学观察,粒－单系祖细胞（CFU－GM）、红细祖细胞（BFU－E）集落培养,细胞表面CD34^ 、CD33^ 、CD3^＋、CD19^＋标记和细胞增周期的动态观察。结果 培养7d时的脐血细胞,CFU－GM、BFU－E集落形成最多,之后逐渐减少,21d时已低于培养前水平。CD34^ 细胞比例在培养后7d达高峰,14d开始下降;CD33^＋细胞比例在培养后7－14d达高峰,21d时开始下降;CD3^ 、C CD19^ 细胞比例均在培养第7d时显著下降,并持续低水平至21d。培养7d后G0期细胞比例明显减少,S期细胞快速增高,至21d时仍有较高比例。培养第21d的细胞涂片见少量中、晚幼粒细胞。结论：理论上脐血干细胞与扩增后的祖细胞混合移植用于成人具有可行性,体外扩增培养7d左右是混合移植的最佳时机。