Ferrero D, Cherasco C, Ortolano B, Giaretta F, Bruno B
Divisione di Ematologia dell'Università di Torino, Azienda Ospedaliera "San Giovanni Battista", Turin, Italy.
Haematologica. 1999 May;84(5):390-6.
All culture systems exploring the early (pre-CFU) hematopoietic compartment are generally complex, time-consuming and unsuitable for routine application. The aim of our study was to develop a stroma-free culture system to quantify early bone marrow (BM) myeloid progenitor cells.
Low density, progenitor cell enriched BM cells underwent a double cytotoxic treatment with CD38 and CD33 monoclonal antibodies + rabbit complement, which depleted 99% of CFU-GM and BFU-E. Then they were cultured, both in agar and in limiting-dilution liquid culture, in the presence of 5637 cell line supernatant (containing GM-CSF, G-CSF and interleukin 1 ), stem cell factor (SCF) and interleukin 3 (IL3).
The largest number (median 14.9 on 1x10(5) cells) and size (>50,000 cells) of myelomonocytic cell clones from CD33Eth /CD38Eth progenitors was reached after 3-4 weeks of liquid culture. SCF, but not IL3, was essential for that growth. The frequency of CD33-/ CD38- progenitors grown in liquid culture was approximately three times greater than the LTC-IC frequency in the same cell suspension. An average 93% of CD33-/CD38- progenitors displayed HLA-DR antigens and 43% generated secondary CFU-GM. In the BM of 9/10 patients, previously exposed to chemotherapy, CD33-/CD38- progenitor frequency was quite low (median 0.9 on 1x10(5) cells), in spite of normal cellularity and morphology and sustained disease remission.
CD33-/CD38- progenitors can be grown and quantified in a stroma-free culture system in a relatively short time. The test can reveal long-lasting, subclinical BM damage induced by chemotherapy and could also be valuable for estimating the amount of early myeloid progenitors for transplantation purposes.
所有探索早期（集落形成单位前）造血区室的培养系统通常都很复杂、耗时，且不适用于常规应用。我们研究的目的是开发一种无基质培养系统，以定量早期骨髓髓系祖细胞。
低密度、富含祖细胞的骨髓细胞先用CD38和CD33单克隆抗体加兔补体进行双重细胞毒性处理，可去除99%的粒-巨噬细胞集落形成单位（CFU-GM）和爆式红系集落形成单位（BFU-E）。然后在5637细胞系上清液（含粒细胞-巨噬细胞集落刺激因子、粒细胞集落刺激因子和白细胞介素1）、干细胞因子（SCF）和白细胞介素3（IL3）存在的情况下，在琼脂和有限稀释液体培养中进行培养。
液体培养3 - 4周后，CD33Eth /CD38Eth祖细胞产生的髓单核细胞克隆数量最多（每1×10(5)个细胞中位数为14.9）且体积最大（>50,000个细胞）。SCF对该生长至关重要，而IL3并非必需。液体培养中生长的CD33-/ CD38-祖细胞频率约为同一细胞悬液中长期培养启动细胞（LTC-IC）频率的三倍。平均93%的CD33-/CD38-祖细胞表达HLA-DR抗原，43%产生次级CFU-GM。在9/10例曾接受化疗的患者骨髓中，尽管细胞数量和形态正常且疾病持续缓解，但CD33-/CD38-祖细胞频率相当低（每1×10(5)个细胞中位数为0.9）。
CD33-/CD38-祖细胞可在无基质培养系统中在相对较短时间内生长并定量。该检测可揭示化疗诱导的长期亚临床骨髓损伤，对于估计用于移植目的的早期髓系祖细胞数量也可能有价值。
