Primary Culture and Differentiation Potential of Mesenchymal Stem Cells from Gushi Chicken Umbilical Cord

doi:10.13304/j.nykjdb.2022.0297

Abstract

Abstract:

In order to study the biological characteristics and induced differentiation of Gushi chicken umbilical cord mesenchymal stem cells （UCMSCs），the experiments of primary culture， cell and directional differentiation were studied. The umbilical cord tissue was selected from 14 d old Gushi chicken embryo， the Wharton’s jelly was cut into 1 mm³ size after the artery and vein of umbilical cord were removed， and then UCMSCs were isolated and cultured by collagenase digestion， cell morphology was observed， UCMSCs growth curves of P3， P9 and p15 generations were drawn to measure population doubling time and clone formation ability of UCMSCs. The results showed that the morphology of Gushi chicken UCMSCs was long spindle shape， and the growth trend was in line with the law of logistic growth curve， showing a typical S-shape. Immunofluorescence and RT-PCR results showed that Gushi chicken UCMSCs expressed mesenchymal stem cell surface marker genes such as CD29， CD44， CD73， CD90 and CD166， but did not express primitive hematopoietic progenitor cell marker CD34 and pan leukocyte marker CD45. Specific staining and PCR showed that UCMSCs could differentiate into adipocytes，osteocytes and chondrocytes in vitro. This experiment confirmed that UCMSCs isolated from Wharton’s jelly of Gushi chicken umbilical cord had good proliferation and differentiation potential in vitro. Moreover， they had similar biological characteristics to UCMSCs isolated from other species in vivo. The multiple differentiation abilities showed the application prospects of cell transplantation， and the results could provide new potential seed cells for regenerative medicine and tissue engineering.

Key words: Gushi chicken, umbilical cord, mesenchymal stem cell, isolation and culture, induction differentiation

摘要：

为了研究固始鸡脐带间充质干细胞（umbilical cord mesenchymal stem cells，UCMSCs）的生物学特性和诱导分化，对其进行原代培养及定向诱导分化研究。选取14日龄固始鸡鸡胚的脐带组织，剥离脐带动、静脉，将华通胶部分剪成1 mm³大小，采用胶原酶消化法分离细胞并进行原代培养，观察细胞形态，绘制P3、P9、P15代生长曲线并测定群体倍增时间及克隆形成能力。结果表明，固始鸡UCMSCs形态为长梭形，生长趋势符合Logistic生长曲线规律，呈典型的S形；免疫荧光和RT-PCR结果显示，固始鸡UCMSCs表达CD29、CD44、CD73、CD90、CD166等间充质干细胞表面标记物基因，但不表达原始造血祖细胞标志物CD34和泛白细胞标志物CD45；经特异性染色和RT-PCR表明，体外诱导固始鸡UCMSCs可分化为脂肪、成骨和成软骨细胞。从固始鸡脐带华通胶中分离所获得的UCMSCs具有良好的体外增殖能力和分化潜能，与从其他物种体内分离的UCMSCs具有相似的生物学特征，它们的多项分化能力预示着细胞移植的应用前景，可为再生医学和组织工程学提供新的潜在种子细胞。

关键词: 固始鸡, 脐带, 间充质干细胞, 分离培养, 诱导分化

CLC Number:

S831

Yue WU, Yun’an WANG, Ha’nan SONG, Weijun GUAN, Nan LI. Primary Culture and Differentiation Potential of Mesenchymal Stem Cells from Gushi Chicken Umbilical Cord[J]. Journal of Agricultural Science and Technology, 2022, 24(9): 79-87.

吴月, 王育南, 宋哈楠, 关伟军, 李楠. 固始鸡脐带间充质干细胞原代培养及其诱导分化潜能研究[J]. 中国农业科技导报, 2022, 24(9): 79-87.

Figures/Tables 9

Table 1 Table for primer information

基因名称 Gene name	引物序列 Primer sequence （3′-5′）	目的片段长度 Target segment length/bp	退火温度 Annealing temperature/℃
GAPDH	F：GGACGCTGGGATGATGTTCT R：GGGTGGTGCTAAGCGTGTTA	292	58.7
CD29	F：TGAATGACACGCAGGAAGATGGAAG R：AGGCAGGGCTGTTAGGTTCTCC	219	59.3
CD44	F：CCTCCTGGCACTGGCATTGATC R：CACTCCACTCTTCATGTCACCATCC	271	58.9
CD73	F：GGCATCGTTGGCTACACTACACAG R：GCTTGTACCACTGGCACCTTCC	326	54.2
CD90	F：ACCAAGGACAACAGGAAGCACATC R：GGTGTTCTGGATCAAGAGGCTGAAG	258	57.8
CD166	F：ACCAGCAGTGCAATGGACAGTTAC R：GCAACCAGCAGAAGACCGACTAC	294	58.3
CD34	F：GCCACTCGCATCCAGAGAACAC R：GTCAGCATCCCCTTCGCATATCG	326	61.4
CD45	F：GTCATGGTTACTCGCTGTGAGGAAG R：CGTCGGAGTTTGAGAAGGAGATGTG	263	58.7
ACAN	F：TGGTGTTGATGGAAGTGGTG R：GTGGGAAAGCCTGAGGTGAG	336	60.4
SOX9	F：GCTCGAAGGAAGCTGGCTGA R：ATGGCGTTGGGGGAGATGTG	254	59.7
LPL	F：GTGACCAAGGTAGACCAGCCATTC R：TCGCCTGACTTCACTCTGACTCTC	248	54.3
PPAR-γ	F：CGAATGCCACAAGCGGAGAAGG R：CACTGCCTCCACAGAGCGAAAC	330	59.6
collagen type Ⅰ	F：GGTAAGGATGGTCGCAATGGTCTC R：GGGTCAGCAGGGTCTCAATTTGG	256	59.1
osteopontin	F：CCTGACATTCCTAGCAAGAGCCAAG R：CTGTCTCCTCCGTCCACCTCAG	398	58.9

Fig. 1 Cell morphology of Gushi chicken UCMSCs

Fig.2 Growth curves of Gushi chicken UCMSCs at P3， P9， and P15

Fig. 3 Colony forming efficiency of different passagesNote： * indicates significant difference at P < 0.05 level.

Fig. 4 Detection of Gushi chicken UCMSCs markers by immunofluorescence stainingNote： DAPI—Stained nuclei； FITC—Immunofluorescence； Merge—Merged image of DAPI and FITC.

Fig. 5 RT-PCR result of marker genesNote： M shows DL1000 DNA Marker；1～8 show GAPDH， CD29， CD44， CD73， CD90， CD166， CD34， CD45 genes，respectively. ** indicates significant difference compared with GAPDH at P<0.01 level.

Fig. 6 Adipogenic differentiation of Gushi chicken UCMSCs tested by oilred O staining and RT-PCRNote： M shows DL1000 DNA Marker；1 shows GAPDH； 2～3 show LPL and PPAR-γ are positive in the inducted group； 4～5 show LPL and PPAR-γ in the control group.

Fig. 7 Osteoblast differentiation of Gushi chicken UCMSCs by calcium deposits within differentiated osteoblast and RT-PCRNote： M shows DL1000 DNA Marker；1 shows GAPDH； 2～3 show collagen type I and osteopontin are positive in the inducted group； 4～5 show collagen type I and osteopontin are negative in the control group.

Fig. 8 Chondrocyte differentiation of Gushi chicken UCMSCs by allianz staining and RT-PCRNote： M shows DL1000 DNA Marker；1 shows GAPDH； 2～3 show SOX9 and ACAN are positive in the inducted group； 4～5 show SOX9 and ACAN are negative in the control group.

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