Identification of marker molecules for negative screening of pancreatic stem cells

doi:10.3969/j.issn.2095-4344.2013.10.013

Abstract

Abstract:

BACKGROUND: Pancreatic stem/progenitor cells are a multipotent self-renewing population and are the potential surrogates in the cell replacement therapy of diabetes mellitus. However, lack of effective cell markers hampers the identification and characterization of pancreatic stem cells.
OBJECTIVE: To explore whether plant lectins peanut agglutinin (PNA) and phaseolus vulgaris agglutinin (PHA) as well as the heat stable antigen (HSA) act as negative screening markers of pancreatic stem cells.
METHODS: The binding of plant lectins (PNA, PHA) and HSA antibody on the cell surface of adult pancreas, developing pancreas and regenerating pancreas was studied through histochemistry and immunofluorescent staining method. The clustering of pancreatic cells according to negative screening molecules was analyzed through flow cytometry. The cell types got from different gates by fluorescence-activated cell sorting were studied through cell culture and observation.
RESULTS AND CONCLUSION: PNA marked pancreatic acinar cells in embryonic and neonatal pancreata, but they did not mark undifferentiated progenitors and precursors, PHA bound most of the undifferentiated and differentiated embryonic pancreatic cells, and HSA marked differentiating and differentiated cells in embryonic pancreata. In adults, both PNA and PHA marked pancreatic acinar cells and partial ductal cells, but not insular cells. HSA could recognize acinar, ductal, vascular endothelial cells, blood cells, and ductal-like cells in the regenerating pancreata. PNA^-/lowHSA^-/low cells accounted for 1.6% of the total pancreatic cells. Through culturing the population of double low cells, they are rich in smaller, triangular or polygonal epithelial cells, and they could be continuously sub-cultured. Our results indicate that PNA and HSA as negative markers can get rid of most differentiated pancreatic cells and part of non-pancreatic cell types, thus enrich pancreatic stem cells.

Key words: stem cells, stem cell culture and differentiation, plant lectins, peanut agglutinin, phaseolus vulgaris agglutinin, heat stable antigen, pancreatic stem cells, negative screening, marker molecule, fluorescence histochemistry, provincial grants-supported paper, stem cell photographs-containing paper

CLC Number:

R394.2

Feng Rui-cheng, Lang Yan-he, Liang Yang, Wu Ying, Teng Chun-bo. Identification of marker molecules for negative screening of pancreatic stem cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(10): 1785-1792.

Figures/Tables 6

利用PHA对胚胎胰腺染色发现，PHA与E12.5大部分胰腺细胞弱结合；在E14.5，PHA结合的胰腺细胞数量增加；在E17.5，PHA与大多数胰腺细胞强结合；出生后5 d，PHA能与结构清晰可辨的腺泡细胞强烈结合，见图1。早期胰芽的广泛染色结果暗示，PHA不仅能够标记分化的腺泡细胞，也能标记胰腺祖及前体细胞。 HSA在E12.5小鼠胰腺仅能与胰芽部分上皮细胞结合；在E14.5，HSA与胰腺芽基初步分化产生的管状上皮细胞结合；在E17.5和出生后5 d，HSA能与大多数胰腺细胞结合，见图1。因而，HSA对正在分化的胰腺祖细胞、前体和终末分化的腺泡能很好地标记。 2.2 PNA、PHA和HSA对小鼠成体胰腺细胞的标记结果见图2。进一步利用荧光组织化学染色分析了PNA、PHA和HSA对成体小鼠胰腺细胞的标记情况。结果发现，PNA能与腺泡细胞强结合，与部分导管上皮内侧也有结合，但与胰腺组织中的某些区域不能结合，见图2A，B。为了进一步分析PNA不能结合哪些细胞，作者利用细胞世系标志分子与PNA进行了共染色。因子8常被作为血管上皮的特异性标记分子，Sca-1是造血世系细胞的标志性分子，胰岛素能够特异性的标记胰岛细胞。共染结果发现，PNA不能与insulin共染，见图2D，不能与因子8共染，见图2E，也不能与Sca-1共定位，见图2F，表明PNA不能标记胰岛细胞、血管内皮细胞及造血世系干细胞。已有研究发现，胰腺损伤后干/祖细胞被刺激增殖分化，参与胰腺再生[26]。胰腺大部切除后再生过程中能产生新生区，其中包括未分化的胰腺祖细胞[23，26]。为了进一步分析PNA对成体胰腺干/祖细胞的标记情况，作者制作了小鼠胰腺损伤再生模型。结果发现，在损伤再生胰腺中，PNA不但能与正常组织腺泡细胞结合，还能与再生组织中的腺泡细胞结合，见图2C，但不能与未分化细胞结合。上述结果表明，PNA可以作为负向筛选分子排除腺泡及部分导管细胞。与PNA相类似，PHA能与正常及损伤后成体胰腺腺泡细胞结合，也能与部分导管上皮细胞所分泌的蛋白结合，但不能结合胰岛，见图3。HSA能弱标记成体腺泡细胞、导管细胞(尤其是小导管)和部分胰岛细胞，强标记血管及血细胞，见图3。在损伤再生胰腺中，HSA与再生的管状上皮细胞结合，见图3。结合胚胎期的染色结果，HSA在成体胰腺中可以标记大部分分化的胰腺细胞类型，正在分化的祖细胞和前体细胞，及血管和造血世系细胞。"

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