Characteristics of bovine muscle satellite cell from different breeds for efficient production of cultured meat

Abstract

The purpose of this study was comparing in vitro performances of three breeds of donor satellite cells for cultured meat and selecting the optimal donor and providing insight into the selection of donors for cultured meat production. Cattle muscle satellite cells were isolated from the muscle tissue of Hanwoo, Holstein, and Jeju black cattle, and then sorted by fluorescence activated cell sorting (FACS). Regarding proliferation of satellite cells, all three breeds showed similar trends. The myogenic potential, based on PAX7 and MYOD mRNA expression levels, was similar or significantly higher for Holstein than other breeds. When the area, width, and fusion index of the myotube were calculated through immunofluorescence staining of myosin, it was expressed upward for Holstein in all experiments except myotube area at passage 8. In addition, it was confirmed that Holstein's muscle satellite cells showed an upward expression in the amount of gene and protein expression related to myogenic. In the case of gene expression of MYOG, DES, and MYH4 known to play a key role in differentiation into muscles, it was confirmed that Holstein's muscle satellite cells expressed higher levels. CAV3, IGF1 and TNNT1, which contribute to hypertrophy and differentiation of muscle cells, showed high expression in Holstein. Our results suggest using cells from Holstein cattle can increase the efficiency of cultured meat production, compared to Hanwoo and Jeju breeds, because the cells exhibit superior differentiation behavior which would lead to greater yields during the maturation phase of bioprocessing.

Keywords: Cattle satellite cell; Cultured meat; Differentiation; Proliferation.

Fig. 1.. All cattle breed donors can produce satellite cells that are pure.

Representative flow cytometry plots, using forward/side scatter (FSC/SSC respectively) and surface expression of CD29 (APC) and CD56 (PE/Cy7), of unsorted muscle cells from the three breeds of cattles. Fluorescence-activated cell sorting (FACS) is indicated by coloured gates.

Fig. 2.. Proliferation of satellite cells from three breeds of cattle during subculture.

(A) Representative brightfield microscopy images of satellite cell morphology on day 2, day 4, and day 6 for passage 4, (B) passage 8, (C) passage 12 (All images taken at 40×).

Fig. 3.. Proliferation capacity of satellite cells from three breeds of cattle.

(A) Representative immunofluorescence microscopy image of satellite cells from three breeds of cattle on day 6 for passages 4, 8, and 12 of cell proliferation. (B) Brightfield microscopy images on day 6 for passages 4, 8, and 12 of cell proliferation (All images taken at 40×). (C) A graph showing the number of nuclei by fluorescent staining on day 6 for passages 4, 8, and 12 of cell proliferation of satellite cells from three breeds cattle. Data are expressed as mean ± SEM (n = 9).

Fig. 4.. Expression of nuclei and satellite cell marker PAX7 in satellite cells from three breeds of cattle.

(A) Representative immunofluorescence microscopy image of the nuclei and PAX7 of satellite cells from three cattle breeds after confluent proliferation at passage 4, (B) passage 8, (C) passage 12 (All images taken at 40×). (D) Expression of PAX7 genes as measured by RT-qPCR at passages 4, 8, and 12 for each breed. Gene expression was normalized against that in Jeju black cattle on Passage 4. Data are expressed as mean ± SEM (n = 3) with differing letters differ significantly (p < 0.05). RT-qPCR, real-time quantitative polymerase chain reaction.

Fig. 5.. Expression of nuclei and satellite cell marker PAX7 in satellite cells from three breeds of cattle.

(A) Representative immunofluorescence microscopy image of the nuclei and PAX7 of satellite cells from three cattle breeds after confluent proliferation at passage 4, (B) passage 8, (C) passage 12 (all images taken at 40×). (D) Expression of MYOD genes as measured by RT-qPCR at passages 4, 8, and 12 for each breed. Gene expression was normalized against that in Jeju black cattle on Passage 4. Data are expressed as mean ± SEM (n = 3) with differing letters differ significantly (p < 0.05).

Fig. 6.. Differentiation of satellite cells from three breeds of cattle during subculture.

(A) Representative brightfield microscopy images of days 1, 3, and 4 of myogenic differentiation at passage 4, (B) passage 8, (C) passage 12 (all images taken at 40×).

Fig. 7.. Differentiation speed and degree of satellite cells from three breeds of cattle.

(A) Representative immunofluorescence microscopy images of satellite cells from three breeds of cattle at passages 4, 8, and 12. Green = myosin, blue = Hoechst. Myotube width, myotube area, and fusion index were determined with ImageJ. For differentiation analysis, only myosin-positive cells with three or more nuclei were rated as myotubes (all images taken at 40×). (B) By converting the number of myotubes with three or more nuclei to a percentage of all nuclei, fusion indices were computed. (C) Myotube areas were measured using ImageJ program. (D) Myotube widths were measured using ImageJ program. Data are expressed as mean ± SEM (n = 9) with differing letters differ significantly (p < 0.05).

Fig. 8.. Myogenic and muscle specific mrna expression profile of satellite cell from three breeds of cattle.

The expression levels of genes specific for myogenic differentiation were determined by using GAPDH as a housekeeping gene at passages 4 and 8 for each breed. (A) Relative mRNA levels of MYOG, (B) DES, (C) MYH4, (D) CAV3, (E) IGF1, (F) TNNT1. Data are expressed as mean ± SEM (n = 3) with differing letters differ significantly (p < 0.05). GAPDH, glyceraldehyde-3-phosphate dehydrogenase.