Enrichment and Clonal Culture of Progenitor Cells During Mouse Postnatal Liver Development in Mice
Enrichment and Clonal Culture of Progenitor Cells During Mouse Postnatal Liver Development in Mice
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DOI：
10.1053/j.gastro.2009.06.001
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发表时间：
2009-09-01
期刊：
影响因子：
29.4
通讯作者：
Nakauchi, Hiromitsu
中科院分区：
文献类型：
--
作者：
Kamiya, Akihide;Kakinuma, Sei;Nakauchi, Hiromitsu
背景与目的：正常出生后肝脏中存在干细胞和祖细胞。然而，由于缺乏特异性的表面标记物，无法克隆分离或分析来自未损伤肝脏的出生后肝干/祖细胞样细胞（PLSC）。本研究旨在建立一种克隆分选的PLSCs原代培养体系。方法：为了研究PLSC的增殖和分化，使用单细胞培养系统纯化和培养来自未损伤肝脏的非实质细胞亚群。在Rho相关激酶（ROCK）抑制剂Y-27632的存在下，使细胞在野生肝细胞衍生的条件培养基中生长。研究结果：我们鉴定了CD 13和CD 133作为在未损伤肝脏中含有PLSC的群体上表达的标记物，并建立了一个有效的单细胞培养系统来克隆分析PLSC。即使使用条件培养基，PLSC的培养也是困难的，但是Y-27632的添加增加了PLSC细胞增殖。在出生后肝脏发育过程中，非实质细胞中祖细胞的比例下降;然而，在3个月大的小鼠中仍然保留了PLSC群体。建立了来源于正常肝脏中克隆分选细胞的长期培养细胞，并将其称为正常肝脏来源的干细胞样细胞（NLS细胞）。NLS细胞在适当的培养条件下可分化为肝细胞样和胆管细胞样细胞，并在连续克隆分选培养中具有自我更新样活性。CD 13和CD 133在来自胎儿和出生后肝脏的祖细胞上表达，而CD 49 f（整合素α 6亚基）仅在PLSC上强烈表达。结论：这些结果表明，在非造血细胞的CD 13（+）CD 49 f（+）CD 133（+）亚群中存在来自未损伤的出生后肝脏的祖细胞。
BACKGROUND & AIMS: Stem and progenitor cells exist in normal postnatal livers. However, it has not been possible to clonally isolate or analyze postnatal liver stem/progenitor-like cells (PLSCs) derived from noninjured livers because of a lack of specific surface markers. This study aimed to establish a primary culture system for clone-sorted PLSCs. METHODS: To investigate proliferation and differentiation of PLSCs, subpopulations of nonparenchymal cells derived from noninjured livers were purified and cultured using a single-cell culture system. Cells were grown in feral liver cell-derived conditioned medium in the presence of the Rho-associated kinase (ROCK) inhibitor Y-27632. RESULTS: We identified CD13 and CD133 as markers expressed on the PLSC-containing population in noninjured livers and established an efficient single-cell Culture system to clonally analyze PLSCs. Culture of PLSCs is difficult, even using conditioned medium, but the addition of Y-27632 increased PLSC cell proliferation. The proportion of progenitor cells among nonparenchymal cells decreased during postnatal liver development; however, a PLSC population was still preserved in 3-month-old mice. Long-term cultivated cells derived from clone-sorted cells in normal livers were established and were called normal-liver-derived stem-like cells (NLS cells). NLS cells could differentiate into hepatocyte-like and cholangiocyte-like cells under appropriate culture conditions and under-went self-renewal-like activity in serial reclone-sorted culture. CD13 and CD133 were expressed on progenitor cells derived from fetal and postnatal liver, whereas CD49f (integrin alpha 6 subunit) was strongly expressed only on PLSCs. CONCLUSIONS: These results demonstrate the presence of progenitor cells in the CD13(+)CD49f(+)CD133(+) subpopulation of nonhematopoietic cells derived from noninjured postnatal livers.