de Jong M O, Rozemuller H, Kieboom D, Visser J W, Wognum A W, Wagemaker G
Institute of Hematology, Erasmus University Rotterdam, The Netherlands.
Leukemia. 1996 Nov;10(11):1813-22.
To characterize Kit expressing mouse bone marrow (BM) cells, and to determine their contribution to short- and long-term repopulation of the hemopoietic system of irradiated recipients, we have purified Kit+ BM cells by flow cytometry. A high level of Kit expression was detectable on 1-2% of BM cells after staining with biologically active biotinylated Kit ligand (KL) or with anti-Kit antibodies (ACK-2). Compared to unfractionated BM, the Kit+ fractions were enriched for immature hemopoietic cells, as shown by morphological differentiation, in vitro culture, and spleen colony formation. Enrichment of colony-forming cells was higher in biotin-KL+ than ACK-2+ fractions. Colony-forming cells were not found in the Kit- subsets. To study the hemopoietic repopulation capacity of the Kit+ and Kit- cells, serial dilutions of the sorted fractions were transplanted into irradiated mice, and peripheral blood of these recipients was monitored regularly for the presence of donor-derived cells during a 1 year period. Nucleated blood cell repopulation by male donor cells in female recipients was assessed using a Y-chromosome specific DNA probe; erythroid repopulation by normal donor cells in W/Wv recipients was examined flow cytometrically by measuring the forward light scatter of donor- and host-type erythrocytes. A 25- to 100-fold enrichment of long-term repopulating ability in the sorted Kit+ fractions showed that Kit+ cells are capable of reconstitution of circulating erythrocytes and nucleated blood cells after BM transplantation. Transient repopulation of the red blood cell lineage was observed after transplantation of Kit- cells. Detection of donor-derived nucleated cells 1 year after transplantation showed that Kit+ cells contributed to donor-type repopulation of bone marrow, spleen and thymus. Our data demonstrate that isolation of BM cells on the basis of Kit expression is a useful addition to the methods that are commonly applied in stem cell enrichment protocols.
为了鉴定表达Kit的小鼠骨髓（BM）细胞，并确定它们对受辐照受体造血系统短期和长期重建的贡献，我们通过流式细胞术纯化了Kit⁺ BM细胞。用生物活性生物素化的Kit配体（KL）或抗Kit抗体（ACK-2）染色后，在1%-2%的BM细胞上可检测到高水平的Kit表达。与未分级的BM相比，Kit⁺ 组分富含未成熟造血细胞，形态分化、体外培养和脾集落形成均表明了这一点。生物素-KL⁺ 组分中集落形成细胞的富集程度高于ACK-2⁺ 组分。在Kit⁻ 亚群中未发现集落形成细胞。为了研究Kit⁺ 和Kit⁻ 细胞的造血重建能力，将分选组分的系列稀释物移植到受辐照小鼠体内，并在1年期间定期监测这些受体的外周血中是否存在供体来源的细胞。使用Y染色体特异性DNA探针评估雌性受体中雄性供体细胞的有核血细胞重建；通过测量供体型和宿主型红细胞的前向光散射，用流式细胞术检测W/Wv受体中正常供体细胞的红系重建。分选的Kit⁺ 组分中长期重建能力富集了25至100倍，表明Kit⁺ 细胞能够在骨髓移植后重建循环红细胞和有核血细胞。移植Kit⁻ 细胞后观察到红细胞系的短暂重建。移植1年后检测到供体来源的有核细胞，表明Kit⁺ 细胞有助于骨髓、脾脏和胸腺的供体型重建。我们的数据表明，基于Kit表达分离BM细胞是干细胞富集方案中常用方法的有益补充。
