骨髓间充质干细胞在致敏与非致敏BALB/c小鼠造血干细胞移植中的应用

背景：造血干细胞移植对多种疾病具有治疗作用,但其取材不便,且细胞数量受年龄限制等原因,故而应用具有一定局限性。 目的：探索骨髓间充质干细胞在致敏与非致敏BALB/c小鼠造血干细胞移植中的应用价值。 方法：将BALB/c小鼠骨髓细胞在体外进行分离,采用贴壁培养的方法获得间充质干细胞,使用流式细胞仪对细胞表面的分子标记进行检测。应用异基因脾细胞输注方法建立致敏动物模型,用绿色荧光染料标记骨髓间充质干细胞,分别移植到致敏和非致敏的受体小鼠体内,并在移植后的不同时间点对间充质干细胞的归巢情况进行检测。对致敏BALB/c小鼠进行照射预处理,联合应用异基因骨髓细胞与同基因间充质干细胞移植,观察BALB/c小鼠的生存情况。 结果与结论：移植48 h后,间充质干细胞在致敏受体和非致敏受体小鼠分别归巢于脾脏和骨髓。在造血干细胞的移植实验中,致敏BALB/c小鼠接受异基因骨髓细胞与同基因骨髓间充质干细胞联合移植,结果显示致敏BALB/c小鼠全部在移植后12-15 d死亡,生存的中位时间是14 d,而仅接受异基因骨髓细胞移植的致敏BALB/c小鼠的中位生存时间为13 d。说明细胞移植后在致敏受体内间充质干细胞主要归巢为脾脏和骨髓,联合应用间充质干细胞移植对异基因造血干/祖细胞植入致敏受体体内并没有起到有效的促进作用。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

异基因骨髓细胞在致敏模型体内归巢示踪与植入分析

目的: 探讨异基因供者骨髓细胞在致敏模型体内的归巢示踪与植入分析。方法: 以异基因脾细胞输注方法建立致敏的BALB/c小鼠模型,同时取正常BABL/c小鼠作为非致敏模型。致敏或非致敏模型经8 Gy 照射后分别经尾静脉移植1×10⁷ C57BL/6小鼠骨髓细胞。用绿色荧光染料CFSE标记供者骨髓细胞,并分别在移植后不同时点(2 h、12 h及48 h),通过组织细胞悬液动态示踪供者细胞在致敏受者各组织的分布。移植后记录各组的生存情况,每周监测造血重建与骨髓恢复情况。予H-2D^b进行标记移植后受者骨髓细胞,检测供者嵌合百分比。结果: CFSE能标记供者骨髓细胞并用于体内示踪实验。动物体内归巢示踪实验表明,与非致敏组相比,异基因供者骨髓细胞在致敏受者体内的外周血、脾脏及股骨的分布均明显减少。植入分析结果发现,非致敏受者于移植后能长期存活,外周血及骨髓细胞均能迅速恢复;而致敏组中,小鼠均于移植后2周左右全部死亡,生存中位数为13 d,外周血及骨髓细胞均随时间推移呈进行性减少。嵌合分析显示移植后第7 d,非致敏受者与致敏受者的供者骨髓细胞百分比分别为(48.07±4.70)％和(0.77±0.11)％,两者差异显著(P<0.01)。结论: 异基因供者骨髓细胞在致敏受者体内脾脏及股骨等部位被清除,不能有效植入。     

小鼠脐血造血干/祖细胞含量与特性初步观察

目的: 探讨小鼠脐血(umbilicalcordblood, UCB)造血干/祖细胞含量与特性。方法: 采用体外集落培养和流式细胞术检测C57BL/6(H-2^b)小鼠脐血与骨髓(bonemarrow, BM)造血干/祖细胞。结果: 小鼠脐血培养7d粒单系祖细胞(CFU-GM)及早期红系祖细胞(BFU-E)集落产率与骨髓相近, 但14dCFU-GM、多向祖细胞(CFU-GEMM)明显高于后者(P<0.05), 且见含细胞多体积巨大的致密型集落。脐血CD₃₄⁺Sca-1⁺细胞亚群也明显高于骨髓(P<0.05)。结论: 小鼠F脐血富含造血干/祖细胞, 具有高增殖潜能。     

NK细胞在小鼠异基因骨髓移植中的作用

目的:研究自然杀伤(NK)细胞在异基因骨髓移植中对移植物抗宿主病(GVHD)、移植排斥、骨髓植入及造血重建的影响。方法:以近交系小鼠C57/6j(H-2^b)为供鼠、BALB/c(H-2^d)为受鼠,在移植物中增加供者的外周T细胞和/或NK细胞进行异基因骨髓移植,用流式细胞仪检测受鼠的CD₃₄^＋细胞计数和H-2K^b＋细胞表达水平,血细胞自动分析仪检测外周血白细胞计数,并结合临床表现和病理检查,比较不同移植组的存活率、GVHD、植入水平及造血重建等。结果:增加NK细胞组的小鼠存活率显著大于不增加NK细胞组,小鼠出现GVHD的数量少、程度轻,外周血白细胞及骨髓CD₃₄^＋细胞恢复快、H-2K^b＋细胞表达水平高。结论:NK细胞抑制小鼠异基因骨髓移植中的GVHD和移植排斥,促进骨髓植入及造血重建。     

抗CD20单抗在致敏受者造血干细胞移植中应用的实验研究

目的： 寻找促进异基因造血干细胞在致敏受者植入的策略研究,探讨抗CD20单抗在致敏受者造血干细胞移植的作用。方法：分别于移植前第14 d及第7 d予BALB/c小鼠输注C57BL/6小鼠的脾细胞建立致敏模型。实验组于移植前第11 d经尾静脉输注抗CD20单抗（美罗华）2 mg/mouse,对照组于移植前第11 d予输注RPMI-1640培养液 0.2 mL/mouse。于第0 d（移植当天）取部分小鼠分离得血清及脾细胞,并检测供者反应性抗体及CD19＋B细胞;部分小鼠予［60Co］致死量照射,4 h后予1×107 C57BL/6小鼠骨髓细胞进行移植,观察生存情况及血常规恢复情况。结果：实验组与对照组血清细胞毒性指数分别为（37.00±3.46）％和（51.80±3.49）％,差异显著（P<0.01）;2组的脾细胞CD19＋B细胞百分比分别为（17.32±3.02）％和（34.26±2.87）％,差异显著（P<0.01）。照射移植后2组受者均于14 d左右全部死亡,生存中位数分别为第13 d及第11 d,Log-rank检验2组间的差别无显著（P>0.05）。濒死动物血常规结果示三系减少,提示受者死于造血衰竭。结论：抗CD20单抗能杀伤受者B细胞,降低致敏程度,但实验中该单抗并不能有效促进异基因造血干细胞在致敏受者的植入。     

骨髓间质干细胞输注对外周血干细胞移植造血恢复的影响

目的:观察骨髓间质干细胞 (MSCs)输注对外周血干细胞移植 (PBSCT)后造血恢复的影响。方法:去脾小鼠经粒细胞集落刺激因子动员后收获外周血单个核细胞 (PBMC)和扩增培养的骨髓间质干细胞 (MSCs),移植给经放 /化疗预处理的BALB/c小鼠,数量分别为10⁶PBMC(PBSCT组)、10⁴MSCs和10⁶ PBMC(实验1组)、10⁶ MSCs和10⁶ PBMC(实验 2组),观察受体鼠 4周的生存率、骨髓有核细胞 (BMNC)、粒细胞巨噬细胞集落形成单位 (CFU-GM)、成纤维细胞集落形成单位 (CFU-F)、外周血白细胞 (WBC)计数等指标。结果:实验 2组的生存率、BMNC、CFU-GM、CFU-F显著高于PBSCT组,WBC计数恢复较PBSCT组快 (P <0.05);实验1组和PBSCT组比较,WBC计算恢复快,CFU-F产率高 (P <0.05)。结论:骨髓间质干细胞输注有促进外周血干细胞移植造血恢复的作用。     

骨髓腔内途径脐血与间质干细胞共移植对造血重建的实验研究

目的：探讨骨髓腔内输注（IBM）脐血与间质干细胞（MSCs）对大鼠造血重建、骨髓MSCs恢复的影响，并研究供体MSCs植入状态以探讨MSCs的作用机制。方法：BrdU标记F344大鼠骨髓MSCs通过双侧胫骨IBM或尾静脉注射（IV）与胎鼠及新生大鼠外周血（FNPB）共移植Wistar雌鼠。监测受鼠存活状况、造血免疫重建、HSCs植入水平及骨髓MSCs恢复情况，并以免疫荧光法检测受鼠骨髓MSCs的来源。结果：(1)2个共移植组60 d存活率均为100%，单纯FNPB移植组仅为66.7%。(2)共移植组的外周血象、骨髓造血干祖细胞集落产率明显高于单纯FNPB移植组，尤以骨髓腔共移植组最佳。(3)2个共移植组的HSCs植入水平无统计学差异，而骨髓腔共移植组明显高于单纯FNPB移植组（P＜0.05）。(4)30 d时各移植组MSCs的增殖能力未达正常水平，但仍以骨髓腔共移植组的恢复情况最佳（P＜0.05）。(5)仅少部分受体可发现供、受体源性MSCs嵌合。 结论：脐血与MSCs共移植可促进受体骨髓MSCs恢复和造血重建，提高HSCs植入率；IBM途径应用安全，促进造血恢复的作用优于IV途径。     

小鼠胎盘间充质干细胞移植对造血系统自然衰老的影响

背景：造血系统作为人体重要组成部分,随着年龄的增长会出现功能不断衰退。 目的：观察小鼠胎盘间充质干细胞对小鼠造血系统自然衰老的延缓作用。 方法：取孕13.5 d Balb/c小鼠胎盘,制备胎盘间充质干细胞。6月龄Balb/c小鼠48只随机分均为细胞移植组和对照组。细胞移植组尾静脉输注胎盘间充质干细胞,每月1次,共6次;对照组输注生理盐水。第3个月进行外周血象、骨髓有核细胞计数、成纤维细胞集落培养、造血祖细胞集落培养和外源性脾集落形成单位计数检测;第6个月时增加骨髓切片观察、骨髓细胞重建造血能力测定。 结果与结论：细胞移植组小鼠一般情况优于对照组;干预第3个月,细胞移植组的骨髓有核细胞计数、巨噬系祖细胞、巨核系祖细胞多于对照组,其他指标无明显差别;干预第6个月,除外周血象仍无差别外,其他的上述指标均明显高于对照组;观察期内两组的上述指标均随时间延长呈下降趋势,但细胞移植组下降速度明显慢于对照组,差异有显著性意义(P < 0.05)。组织学观察发现,细胞移植组骨髓造血组织较对照组丰富,而对照组骨髓脂肪化显著增加;实验还发现细胞移植组骨髓细胞的造血重建能力明显优于对照组。结果提示,小鼠胎盘间充质干细胞能够延缓小鼠造血系统的衰退。     

促进胚胎干细胞来源造血干/祖细胞移植后细胞免疫功能恢复

目的:体外诱导胚胎干细胞分化为造血干/祖细胞过程中, 增加成熟T淋巴细胞的含量, 以促进其重建致死量照射小鼠的造血功能后免疫功能的早期重建。方法:胚胎干细胞在含甲基纤维素的培养基中自由分化形成胚胎体, 分化第6d添加造血生长因子, 同时添加胸腺肽, 流式细胞仪检测分化细胞中CD₃₄⁺的造血干/祖细胞和CD⁺₃的成熟T淋巴细胞含量, 最后将分化细胞注射入致死量照射小鼠体内, 观察60d, 以移植物抗宿主病(GVHD)发病率作为T淋巴细胞免疫功能的指标, 用PCR检测Sry反映移植细胞在宿主体内的存活。结果:分化第13d, 未加胸腺肽, CD⁺₃的成熟T淋巴细胞含量仅10.52%, 重建造血后无GVHD发生;添加胸腺肽, CD⁺₃的成熟T淋巴细胞含量升高达22.93%, 重建造血后GVHD发病率100%。结论:胚胎干细胞体外分化为造血干/祖细胞过程中, 添加胸腺肽, 能增加CD⁺₃的成熟T淋巴细胞含量, 体内重建造血后细胞免疫功能恢复较快。     

表达SDF-1/HOXB4融合基因的间充质干细胞联合脐血CD34~+干细胞共移植对辐射损伤小鼠的影响

目的观察表达SDF-1/HOXB4融合基因的间充质干细胞(mesenchymal stem cells,MSCs)联合脐血CD34+造血干细胞(hematopoietic stem cells,HSCs)共移植对辐射损伤小鼠的影响。方法表达SDF-1、HOXB4和SDF-1/HOXB4基因的3个腺病毒载体分别转染正常人骨髓MSCs,将其联合脐血CD34+细胞经尾静脉移植到辐射损伤的NOD/SCID小鼠体内(MSCs 8×105细胞/只,CD34+1×105细胞/只),分别为SDF-1/MSCs+CD34+组(SDF-1组)、HOXB4/MSCs+CD34+组(HOXB4组)、SDF1-HOXB4/MSCs+CD34+组(S-H组),另外3组为未转染MSCs+CD34+组(MSC-HSC组)、单纯CD34+组(HSC组)、单纯辐照组(IR组)。检测移植后各组小鼠存活率、外周血象恢复、骨髓病理变化及人源CD45+细胞植入率。结果 S-H组小鼠存活率高,且外周血WBC、HGB、PLT和骨髓造血功能恢复快。在移植后6周骨髓CD45+细胞植入率(47.43%±8.89%)较其余各组高。结论表达SDF-1/HOXB4融合基因的间充质干细胞(MSCs)联合脐血CD34+造血干细胞(HSCs)共移植能促进造血重建及植入,提高移植成功率。     

骨髓间充质干细胞和心脏Sca1阳性细胞移植治疗心肌梗死的效果比较及其机制研究

目的探讨骨髓来源的间充质干细胞(MSCs)和心脏来源的Sca1阳性干细胞移植对小鼠急性心肌梗死后的心功能恢复的影响。方法分别从成年C57BL/C小鼠的骨髓分离获取骨髓间充质干细胞,从心脏利用流式分选获取Sca1阳性干细胞,并在体外进行培养、扩增。C57BL/C小鼠雄性小鼠24只,按照以下方式分组,每组6只,假手术组,生理盐水对照组,MSCs治疗组和Sca1阳性细胞治疗组。小鼠麻醉后开胸,利用结扎心脏冠状动脉的左前降支建立急性心肌梗死模型,建模成功后分别在梗死区周围心肌内注射上述细胞或者生理盐水,假手术组小鼠不结扎冠状动脉。4周后,利用心脏超声观察小鼠左心室射血分数(LVEF)以及左心室舒张末期容积,以此评估心功能改变。细胞培养至第4代,采用荧光半定量PCR的方式分析细胞表达血管内皮生长因子(VEGFa和VEGFb)的情况。结果分离培养的骨髓MSCs贴壁生长,呈梭形;Sca1阳性干细胞在心脏组织的阳性率约为18%,培养后细胞贴壁生长,成短梭形,形态较MSCs更饱满。在细胞移植治疗后,与Sca1阳性干细胞治疗相比,MSCs治疗可以增加心梗后小鼠的存活率。细胞移植治疗4周后,骨髓来源MSCs治疗组的LVEF明显高于心脏来源Sca1阳性干细胞,且更有利于减少心梗后左心室舒张末期容积的扩大,改善心肌重塑。骨髓来源的MSCs表达VEGFa和VEGFb明显高于Sca1阳性细胞。结论心脏来源的Sca1阳性干细胞和骨髓来源的MSCs均可提高LVEF,促进心功能的恢复,且后者优于前者,这可能与MSCs高表达VEGF有关。     

Cotransplanted bone marrow derived mesenchymal stem cells (MSC) enhanced engraftment of hematopoietic stem cells in a MSC-dose dependent manner in NOD/SCID mice

Transplantation of marrow-derived mesenchymal stem cells (MSCs), expanded by culture in addition to whole bone marrow, has been shown to enhance engraftment of human hematopoietic stem cells (HSCs). Our hypothesis was that there might be an optimum ratio range that could enhance engraftment. We examined the percent donor chimerism according to the ratio of HSCs to MSCs in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. We tested a series of ratios of co-transplanted CD34(+) -selected bone marrow cells, and marrow-derived MSCs into sublethally irradiated NOD/SCID mice. In all experiments, 1x10(5) bone marrow derived human CD34(+) cells were administered to each mouse and human MSCs from different donors were infused concomitantly. We repeated the procedure three times and evaluated engraftment with flow cytometry four weeks after each transplantation. Serial ratios of HSCs to MSCs were 1:0, 1:1, 1:2 and 1:4, in the first experiment, 1:0, 1:1, 1:2, 1:4 and 1:8 in the second and 1:0, 1:1, 1:4, 1:8 and 1:16 in the third. Cotransplantation of HSCs and MSCs enhanced engraftment as the dose of MSCs increased. Our results suggest that the optimal ratio of HSCs and MSCs for cotransplantation might be in the range of 1:8-1:16; whereas, an excessive dose of MSCs might decrease engraftment efficiency.     

致敏小鼠骨髓间充质干细胞体外培养及多向分化功能的测定

目的：评价异基因脾细胞输注致敏的小鼠骨髓源性间充质干细胞(MSCs)的体外培养生长能力及其多向分化功能。方法：应用贴壁培养法体外培养间充质干细胞,流式检测其表面标志以及检测其成骨、成脂和成肌多向分化状况;结果：致敏小鼠骨髓源性MSCs与非致敏小鼠骨髓源性MSCs比较,形态学无差异且均表达CD29+、CD105+、CD44+和Sca-1+ ;CD34-、CD11b-;同时在相应的诱导条件下具有向成骨、成脂、成肌多向分化的能力。结论：异基因脾细胞输注致敏的小鼠,其骨髓源性MSCs的形态学和功能与正常小鼠的MSCs比较评估未见异常。     

Cotransplantation of HLA-identical sibling culture-expanded mesenchymal stem cells and hematopoietic stem cells in hematologic malignancy patients.

Mesenchymal stem cells (MSCs) are found in a variety of tissues, including human bone marrow; secrete hematopoietic cytokines; support hematopoietic progenitors in vitro; and possess potent immunosuppressive properties. We hypothesized that cotransplantation of culture-expanded MSCs and hematopoietic stem cells (HSCs) from HLA-identical sibling donors after myeloablative therapy could facilitate engraftment and lessen graft-versus-host disease (GVHD); however, the safety and feasibility of this approach needed to be established. In an open-label, multicenter trial, we coadministered culture-expanded MSCs with HLA-identical sibling-matched HSCs in hematologic malignancy patients. Patients received either bone marrow or peripheral blood stem cells as the HSC source. Patients received 1 of 4 study-specified transplant conditioning regimens and methotrexate (days 1, 3, and 6) and cyclosporine as GVHD prophylaxis. On day 0, patients were given culture-expanded MSCs intravenously (1.0-5.0 x 10(6)/kg) 4 hours before infusion of either bone marrow or peripheral blood stem cells. Forty-six patients (median age, 44.5 years; range, 19-61 years) received MSCs and HLA-matched sibling allografts. MSC infusions were well tolerated, without any infusion-related adverse events. The median times to neutrophil (absolute neutrophil count > or = 0.500 x 10(9)/L) and platelet (platelet count > or = 20 x 10(9)/L) engraftment were 14.0 days (range, 11.0-26.0 days) and 20 days (range, 15.0-36.0 days), respectively. Grade II to IV acute GVHD was observed in 13 (28%) of 46 patients. Chronic GVHD was observed in 22 (61%) of 36 patients who survived at least 90 days; it was extensive in 8 patients. Eleven patients (24%) experienced relapse at a median time to progression of 213.5 days (range, 14-688 days). The probability of patients attaining disease- or progression-free survival at 2 years after MSC infusion was 53%. Cotransplantation of HLA-identical sibling culture-expanded MSCs with an HLA-identical sibling HSC transplant is feasible and seems to be safe, without immediate infusional or late MSC-associated toxicities. The optimal MSC dose and frequency of administration to prevent or treat GVHD during allogeneic HSC transplantation should be evaluated further in phase II clinical trials.     

非黏附骨髓源干细胞治疗急性放射损伤

背景：目前对于辐射剂量超过8 Gy的急性放射病尚缺乏有效的治疗方案,间充质干细胞可以分泌多种造血因子、重建造血,在放射损伤救治中具有重要意义。 目的：探讨非黏附骨髓源干细胞在8.5 Gy X射线照射所致急性骨髓型放射损伤救治中的作用及作用机制。 方法：取胎儿四肢长骨的非黏附骨髓源干细胞,分析其麦面抗原,细胞周期,成骨和成脂分化潜能,以及血管内皮生长因子及Annexin A2表达。BALB/C小鼠受8.5 Gy一次性全身均匀X射线照射后随机分成骨髓源干细胞组和对照组,骨髓源干细胞组小鼠在X射线照射2 h内经尾静脉输注含3×106 CFDA-SE标记的人非黏附骨髓源干细胞的细胞悬液0.3 mL,对照组小鼠在X射线照射2 h内输注0.3 mL生理盐水。观察骨髓源干细胞的分布情况、小鼠的存活率、白细胞变化、骨髓病理变化及骨髓中新生血管形成情况。 结果与结论：X射线照射后移植的非黏附间充质干细胞可以向损伤部位归巢;骨髓源干细胞组小鼠存活率明显高于对照组;与对照组相比,骨髓源干细胞组小鼠外周血白细胞计数下降慢且恢复迅速,X射线照射后14 d左右达最低,30 d基本恢复至正常水平。X射线照射后21 d,骨髓源干细胞组骨髓增生活跃,骨髓腔内新生造血灶显著多于对照,血管密度亦显著高于对照组。说明人胎儿非黏附骨髓源干细胞促进急性放射损伤小鼠骨髓内新生血管形成,改善并加快受损小鼠造血功能的恢复。     

Co-transplantation of human mesenchymal stem cells promotes human CD34+ cells engraftment in a dose-dependent fashion in NOD/SCID mice

Mesenchymal stem cells (MSCs) have recently been identified and characterized in humans. Moreover, MSC secrete cytokines that can support hematopoietic progenitor growth. In the present study, we evaluated whether the efficacy of hematopoietic stem cell transplantation is improved by their co-transplantation with MSC, and whether this is positively correlated with the dose of infused MSCs. Accordingly, irradiated NOD/SCID mice were transplanted with 1 x 10(5) human CD34+ cells in the presence or absence of culture expanded MSCs (1 x 10(6) or 5 x 10(6)). We evaluated human hematopoietic cell engraftment by flow cytometry and assessed MSC tissue distributions by fluorescence in situ hybridization. We found that CD45+ and CD34+ cell levels were significantly elevated in a dose-dependent manner in co-transplanted mice 4 weeks after transplantation. The engraftments of CD33+ and CD19+ cells also increased dose-dependently. However, the engraftment of CD3+ cells did not increase after co-transplantation with MSCs. Human Y chromosome+ cells were observed in multiple tissues and were more frequently observed in mice co-transplanted with 5 x 10(6) rather than 1 x 10(6) MSCs. These results suggest that MSCs are capable of enhancing hematopoietic cell engraftment and distribution in multiple organs in a dose-dependent fashion.     

Reduction of marrow hematopoietic progenitor and stem cell content is not sufficient for enhanced syngeneic engraftment

The mechanisms regulating long-term engraftment of primitive stem cells are largely unknown. Most conditioning strategies use myeloablative agents for experimental or clinical hematopoietic stem cell transplantation. Host conditioning regimens, in part, have been designed on the assumption that transplanted cells home to specific marrow sites and if these sites are occupied by host stem cells, engraftment will not take place. However, there is now evidence that stable long-term syngeneic engraftment may occur in the absence of host marrow stem cell depletion. To further study the association of engraftment with stem cell depletion, we investigated whether the marked egress of hematopoietic progenitor and stem cells from the marrow into the peripheral blood in C57BL6 mice following a single dose of cyclophosphamide (day 1) and four days of G-CSF (days 3-6) afforded an increased opportunity for long-term syngeneic donor engraftment. During and after mobilization, glucose phosphate isomerase (GPI)-1(b) mice received 30 x 10(6) GPI-1(a) marrow cells without further myeloablation. The level of donor/recipient chimerism was assessed in cell lysates after six months. Increased long-term syngeneic donor engraftment was observed prior to mobilization (before day 6), during a period of active hematopoietic regeneration following the administration of cyclophosphamide. Hematopoietic regeneration was evidenced by a reduced but rapidly increasing marrow cellularity and an increased proportion of hematopoietic progenitors in S-phase. In contrast, long-term syngeneic donor engraftment was not increased over controls during the period of maximum progenitor and stem cell mobilization (after day 5). At this time there were minimal numbers of progenitor and stem cells in the marrow. These data suggest that in the absence of host stem cell ablation, maximal engraftment does not occur during marrow progenitor or stem cell depletion, suggesting that the presence of "open" marrow sites is not a prerequisite for engraftment. The mechanisms for increased engraftment during progenitor cell regeneration following cyclophosphamide need further investigation. Understanding the mechanisms for engraftment without host stem cell ablation may allow strategies for improved long-term engraftment of syngeneic or autologous stem cells with reduced post-transplant toxicity.