Cell Biology and Neuroscience, Istituto Superiore di Sanita', Rome, Italy.
Blood Cells Mol Dis. 2011 Oct 15;47(3):182-97. doi: 10.1016/j.bcmd.2011.06.001. Epub 2011 Jul 20.
The number of erythroblasts generated ex-vivo under human-erythroid massive-amplification conditions by mononuclear cells from one unit of adult blood (10(10)) are insufficient for transfusion (10(12) red cells), emphasizing the need for studies to characterize cellular interactions during culture to increase erythroblast production. To identify the cell populations which generate erythroblasts under human-erythroid-massive-amplification conditions and the factors that limit proliferation, day 10 non-erythroblasts and immature- and mature-erythroblasts were separated by sorting, labelled with carboxyfluorescein-diacetate-succinimidyl-ester and re-cultured either under these conditions (for proliferation, maturation and/or apoptosis/autophagy determinations) or in semisolid media (for progenitor cell determination). Non-erythroblasts contained 54% of the progenitor cells but did not grow under human-erythroid-massive-amplification conditions. Immature-erythroblasts contained 25% of the progenitor cells and generated erythroblasts under human-erythroid-massive-amplification conditions (FI at 48 h=2.57±1.15). Mature-erythroblasts did not generate colonies and died in human-erythroid-massive-amplification conditions. In sequential sorting/re-culture experiments, immature-erythroblasts retained the ability to generate erythroblasts for 6 days and generated 2-5-fold more cells than the corresponding unfractionated population, suggesting that mature-erythroblasts may limit erythroblast expansion. In co-cultures of carboxyfluorescein-diacetate-succinimidyl-ester-labelled-immature-erythroblasts with mature-erythroblasts at increasing ratios, cell numbers did not increase and proliferation, maturation and apoptotic rates were unchanged. However, Acridine Orange staining (a marker for autophagic death) increased from ~3.2% in cultures with immature-erythroblasts alone to 14-22% in cultures of mature-erythroblasts with and without immature-erythroblasts. In conclusion, these data identify immature-erythroblasts as the cells that generate additional erythroblasts in human-erythroid-massive-amplification cultures and autophagy as the leading cause of death limiting the final cellular output of these cultures.
在人类红细胞大量扩增条件下，由单个成人血液单位（约 10(10)）的单核细胞产生的红细胞数量不足以用于输血（约 10(12)个红细胞），这强调了需要研究在培养过程中表征细胞相互作用以增加红细胞生成的必要性。为了确定在人类红细胞大量扩增条件下产生红细胞的细胞群体以及限制增殖的因素，第 10 天的非红细胞、未成熟和成熟红细胞通过分选进行分离，用羧基荧光素二乙酸琥珀酰亚胺酯进行标记，并在这些条件下（用于增殖、成熟和/或凋亡/自噬测定）或在半固体培养基中（用于祖细胞测定）再培养。非红细胞含有 54%的祖细胞，但在人类红细胞大量扩增条件下不生长。未成熟红细胞含有 25%的祖细胞，并在人类红细胞大量扩增条件下产生红细胞（48 小时时的 FI 为 2.57±1.15）。成熟红细胞在人类红细胞大量扩增条件下不产生集落并死亡。在连续分选/再培养实验中，未成熟红细胞在 6 天内保留了产生红细胞的能力，并比相应的未分选群体产生 2-5 倍的细胞，这表明成熟红细胞可能限制红细胞的扩增。在未成熟红细胞与成熟红细胞以逐渐增加的比例共培养时，细胞数量没有增加，增殖、成熟和凋亡率没有变化。然而，吖啶橙染色（自噬死亡的标志物）从单独培养未成熟红细胞时的约 3.2%增加到有和没有未成熟红细胞的成熟红细胞共培养时的 14-22%。总之，这些数据表明，未成熟红细胞是在人类红细胞大量扩增培养中产生额外红细胞的细胞，自噬是限制这些培养物最终细胞产量的主要死亡原因。
