Scientia Agricultura Sinica ›› 2022, Vol. 55 ›› Issue (22): 4500-4512.doi: 10.3864/j.issn.0578-1752.2022.22.014

• FOOD SCIENCE AND ENGINEERING • Previous Articles     Next Articles

Differentiation of Porcine Muscle Stem Cells in Three-Dimensional Hydrogels

CHEN Yu(),ZHU HaoZhe,CHEN YiChun,LIU Zheng,DING Xi,GUO Yun,DING ShiJie(),ZHOU GuangHong()   

  1. College of Food Science and Technology, Nanjing Agricultural University/National Meat Quality and Safety Control Engineering Technology Research Center/Key Laboratory of Meat Processing and Quality Control, Ministry of Education, Nanjing 210095
  • Received:2022-03-03 Accepted:2022-06-16 Online:2022-11-16 Published:2022-12-14
  • Contact: ShiJie DING,GuangHong ZHOU E-mail:2020808131@stu.njau.edu.cn;shijieding@njau.edu.cn;zhou@hotmail.com

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Abstract:

【Objective】 The objective of this study was to explore the differentiation effect of porcine muscle stem cells in three-dimensional hydrogels, and to provide a guidance for inducing muscle stem cells to differentiate into muscle tissue in vitro.【Method】 Some porcine muscle stem cells were respectively induced to differentiate under the conditions of 2D and 3D (2D condition means culturing cells in culture dishes; 3D condition means culturing cells in hydrogels). The RNA and protein samples of porcine muscle stem cells cultured in 2D were collected at proliferation, pre-differentiation, early differentiation, mature differentiation, and late differentiation, respectively, and those in 3D were also collected at day 7 and day 14 of differentiation, respectively. Then, RT-qPCR was used to compare the expression levels of the myogenic-related genes, including the genes of MYOG, CAV-3, MyHC-slow, and MyHC-2a, under 2D and 3D differentiation conditions. Correspondingly, the Western Blot was used to detect the expression levels of MyHC protein and MYOG protein in the two conditions. Moreover, the immunofluorescence staining was used to observe the myotubes formed in cell culture dishes and hydrogels. Further, the amino acid content and composition of the cultured muscle tissue were analyzed by an amino acid automatic analyzer at day 14 of differentiation. 【Result】 The porcine muscle stem cells started to fuse to form myotubes at day 3 of differentiation in 2D. The myotubes formed in 2D matured at day 7 and divorced from culture dish afterwards. The porcine muscle stem cells were still globe and had low expression of MYOG and CAV-3 at day 7 of differentiation in 3D. Multinucleated myotubes formed at day 14 and the expression of MYOG and CAV-3 reached levels of 2D differentiation. The cells in hydrogels had higher expression of terminal differentiation genes MyHC-slow and MyHC-2a than the cells in culture dishes. The expression of MyHC-slow was 12 times that at day 7 in 2D and the expression of MyHC-2a was 4 times that at day7 in 2D, but the expression of MyHC protein was only 1/6 that at day 7 in 2D. Amino acid analysis results showed that the contents of 17 hydrolyzed amino acids in cultured muscle tissue were all lower than those in pork, and the ratio of essential amino acids was also lower in cultured muscle tissue, but the ratio of flavor amino acids was higher. 【Conclusion】 The porcine muscle stem cells could differentiate into myotubes in 3D collagen hydrogels in vitro, and the 3D condition was positive to the expression of myogenic differentiation related genes, but further research was needed to achieve high expression of MyHC protein. The flavor amino acid content of the muscle tissue cultured in this way was high, which might mean good flavor.

Key words: porcine muscle stem cells, differentiation, hydrogels, myotubes, cultured meat

Table 1

The primer information of RT-qPCR"

基因
Gene 		引物序列
Primers sequence
MYOG 上游 Forward AACCCCACTTCTATGACGGG
下游 Reverse TTATCTTCCAGGGGCACTCG
MyHC-slow 上游 Forward CCGTGCTCCGTCTTCTTTCC
下游 Reverse CGCTCCTTCTCTGACTTGCG
MyHC-2a 上游 Forward AGGACCAAGTACGAGACGGA
下游 Reverse AGCTTCCACGTGTTCCTCAG
CAV-3 上游 Forward GCCCAGATCGTCAAGGACAT
下游 Reverse CAGGCGGTAGCACCAATACT
GAPDH 上游 Forward GTCGGAGTGAACGGATTTGGC
下游 Reverse CTTGCCGTGGGTGGAATCAT

Fig. 1

The morphology of porcine muscle stem cells differentiated for different days (40×) A: Porcine muscle stem cells in proliferation (Myoblast); B: Differentiated for 0 day (pre-differentiation); C: Differentiated for 3 days; D: Differentiated for 4 days; E: Differentiated for 7 days; F: Differentiated for 8 days"

Fig. 2

The morphology of cultured muscle tissue in mold for different days"

Fig. 3

Relative area changes of hydrogels"

Fig. 4

The immunofluorescence staining of porcine muscle stem cells differentiated for different days (100×) A: Porcine muscle stem cells in proliferation (Myoblast); B: Differentiated for 0 days (2D); C: Differentiated for 3 days (2D); D: Differentiated for 7 days (2D); E: Differentiated for 8 days (2D); F: Differentiated for 7 days (3D); G: Differentiated for 14 days (3D)"

Table 2

Contents of amino acids in pork and cultured meat (mg?g-1)"

名称
Name		 猪肉
Pork		 培养肌肉组织
Cultured muscle tissue
必需氨基酸 EAA
苏氨酸Thr 9.53 1.95
缬氨酸Val 11.42 1.64
蛋氨酸Met 2.34 0.04
异亮氨酸Ile 10.18 1.14
亮氨酸Leu 16.75 2.58
苯丙氨酸Phe* 9.42 1.68
赖氨酸Lys 13.41 1.46
非必需氨基酸 NEAA
天冬氨酸Asp* 15.52 1.72
丝氨酸Ser 8.12 3.35
谷氨酸Glu* 33.47 9.46
甘氨酸Gly* 8.96 6.17
丙氨酸Ala* 10.85 3.90
半胱氨酸Cys 2.86 0.20
酪氨酸Tyr* 9.15 0.92
组氨酸His 9.39 0.82
精氨酸Arg 14.96 4.17
脯氨酸Pro 10.47 3.65
必需氨基酸 EAA 73.05 10.49
非必需氨基酸 NEAA 123.75 34.35
呈味氨基酸 FAA 97.37 23.85
总氨基酸 TAA 196.80 44.84
FAA/TAA 44.39% 53.19%
EAA/TAA 37.12% 23.39%
*为呈味氨基酸 *Refers to flavoring amino acids

Fig. 5

The changes of the key genes during differentiation of porcine muscle stem cells Myoblast: Porcine muscle stem cells in proliferation; pre-diff: Differentiated for 0 days (2D); 2D-D3: Differentiated for 3 days (2D); 2D-D7: Differentiated for 7 days (2D); 2D-D8: Differentiated for 8 days (2D); 3D-D7: Differentiated for 7 days (3D); 3D-D14: Differentiated for 14 days (3D). The same as"

Fig. 6

The changes of the key protein during differentiation of porcine muscle stem cells"

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