Isolation and Characterization of Tissue Resident CD29-Positive Progenitor Cells in Livestock to Generate a Three-Dimensional Meat Bud

Naraoka, Yuna; Mabuchi, Yo; Yoneyama, Yoshimasa; Suto, Eriko Grace; Hisamatsu, Daisuke; Ikeda, Mami; Ito, Rieko; Nakamura, Toshio; Takebe, Takanori; Akazawa, Chihiro

doi:10.3390/cells10092499

Cited by 13 publications

(15 citation statements)

References 20 publications

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“…First, we confirmed the cross-reactivity between cattle and antigen species based on previous reports [11][12][13], as the number of monoclonal antibodies targeting bovine antigens is limited. Notably, we used bovine muscle rather than bovine adipose to confirm crossreactivity.…”

Section: Isolation and Classification Of Aspcssupporting

confidence: 81%

Conservation of Markers and Stemness in Adipose Stem and Progenitor Cells between Cattle and Other Species

Ishida

Mabuchi

Naraoka

et al. 2023

IJMS

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Adipose stem and progenitor cells (ASPCs) have been isolated from humans and animals for use in regenerative medicine and therapy. However, knowledge of ASPCs in other species is limited. Particularly, ASPCs in livestock are expected to enhance the fat content and meat composition. In this study, we isolated bovine ASPCs using cell surface markers. Specifically, we focused on ASPC markers in humans and experimental animals, namely CD26, CD146, and CD54. Stromal vascular fraction cells from bovine fat were separated using flow cytometry before primary culture. We evaluated the self-renewal and adipogenic potential of each fraction. We identified four cell populations: CD26−CD146+CD54+, CD26−CD146+CD54−, CD26−CD146−, and CD26+CD146−. Among them, the CD26−CD146+ fraction, particularly CD54+, demonstrated the properties of preadipocytes (PreAs), characterized by slow proliferation and a high adipogenic capacity. In conclusion, we could collect and characterize possible PreAs as CD26−CD146+CD54+ or CD26−CD146+CD54−, which are expected for in vitro bovine adipogenic assays in the future.

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Section: Isolation and Classification Of Aspcssupporting

confidence: 81%

Conservation of Markers and Stemness in Adipose Stem and Progenitor Cells between Cattle and Other Species

Ishida

Mabuchi

Naraoka

et al. 2023

IJMS

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“…A multi-color flow cytometry protocol enables to simultaneously evaluate the expression of different markers and as such to identify MSC heterogeneity [56,57]. However, there are currently no specific criteria to immunophenotype bovine MSCs, partly due to the limited availability of bovine-specific monoclonal antibodies (mAbs) or validated cross-reactive mAbs [9], as illustrated by the study of Naraoka et al Indeed, half of the tested 246 Abs showed cross-reactivity toward bovine epitopes, but 5%-10% was the highest percentage of cross-reactivity reported (for 97 Abs) while only 1%-5% cross-reactivity was reported for 17 Abs [58].…”

Section: Introductionmentioning

confidence: 99%

Validation of multiparametric panels for bovine mesenchymal stromal cell phenotyping

et al. 2023

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Bovine mesenchymal stromal cells (MSCs) display important features that render them valuable for cell therapy and tissue engineering strategies, such as self‐renewal, multi‐lineage differentiation, as well as immunomodulatory properties. These cells are also promising candidates to produce cultured meat. For all these applications, it is imperative to unequivocally identify this cell population. The isolation and in vitro tri‐lineage differentiation of bovine MSCs is already described, but data on their immunophenotypic characterization is not yet complete. The currently limited availability of monoclonal antibodies (mAbs) specific for bovine MSC markers strongly hampers this research. Following the minimal criteria defined for human MSCs, bovine MSCs should express CD73, CD90, and CD105 and lack expression of CD14 or CD11b, CD34, CD45, CD79α, or CD19, and MHC‐II. Additional surface proteins which have been reported to be expressed include CD29, CD44, and CD106. In this study, we aimed to immunophenotype bovine adipose tissue (AT)‐derived MSCs using multi‐color flow cytometry. To this end, 13 commercial Abs were screened for recognizing bovine epitopes using the appropriate positive controls. Using flow cytometry and immunofluorescence microscopy, cross‐reactivity was confirmed for CD34, CD73, CD79α, and CD90. Unfortunately, none of the evaluated CD105 and CD106 Abs cross‐reacted with bovine cells. Subsequently, AT‐derived bovine MSCs were characterized using multi‐color flow cytometry based on their expression of nine markers. Bovine MSCs clearly expressed CD29 and CD44, and lacked expression of CD14, CD45, CD73, CD79α, and MHCII, while a variable expression was observed for CD34 and CD90. In addition, the mRNA transcription level of different markers was analyzed using reverse transcription quantitative polymerase chain reaction. Using these panels, bovine MSCs can be properly immunophenotyped which allows a better characterization of this heterogenous cell population.

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“…There are several commercially available cell freezing media (Yokonishi et al, 2014;Balci-Hayta et al, 2018;Fu et al, 2020;Naraoka et al, 2021). In this study, three representative media were selected to compare the effect of cryopreservation.…”

Section: Discussionmentioning

confidence: 99%

“…This is important to increase the versatility of our result by avoiding the use of specialized equipment such as a liquid nitrogen tank (Miyamoto et al, 2018). In this study, three commercially available freezing media (CB1, CB2, and LB1) were used as representative media (Yokonishi et al, 2014;Jang et al, 2017;Balci-Hayta et al, 2018;Naraoka et al, 2021). CB1 and LB1 from two different companies have been widely used to cryopreserve various kinds of mammalian cells, including bovine cells.…”

Section: E Ect Of Cell Freezing Media On Proliferation Ability Of Bov...mentioning

confidence: 99%

Repeated and long-term cryopreservation of primary bovine myogenic cells to maintain quality in biomimetic cultured meat

Kakehi

Yoshida²,

Takahashi³

et al. 2023

Front. Sustain. Food Syst.

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Cultured meat produced using cell culture technology can potentially alleviate many of the ethical, environmental, and public health concerns associated with conventional livestock meat production. The industrialization of cultured meat for wide-spread adoption requires new methods to efficiently collect high-quality cells and to preserve their cell quality. Cryopreservation is a widely used technique to enable the long-term storage of cells without causing severe damage. In this study, we focused on the feasibility of cryopreservation to maintain cell quality for storage of bovine myogenic cells harvested from bovine meat based on our unique primary culture method. Primary bovine cells were incubated in a culture dish and then cryopreserved at −80°C for 1 week or 1 year. After thawing, the cells were further cultured for several passages to evaluate the abilities of the cells to proliferate or differentiate into myotubes. Furthermore, the cells were repeatedly cryopreserved for 1 week each time to investigate the impact of the repeated freezing and thawing. Consequently, long-term (within 1 year) or repeated (up to 3 times for 1 week each) cryopreservation at −80°C caused no degradation in the abilities of the cells to proliferate or differentiate, which is important for cultured meat production. We also confirmed that the cryopreservation did not require any unique cell freezing media. Moreover, based on our tissue engineering technique, our cryopreserved bovine myogenic cells had the ability to form sarcomere structures and produce muscle contractions even after they were frozen for 1 year. Although the bovine muscle tissues described here require more mature structures and functions in order to closely mimic native muscle tissue, we believe that the functional maturation of myogenic cells is essential to produce a “tissue-engineered meat” that will have native-like nutrients, texture, and taste that consumers will expect in the future. These results reveal the potential of cryopreserving quality-controlled bovine myogenic cells to contribute to a stable supply of high-quality cells for cultured meat production.

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Isolation and Characterization of Tissue Resident CD29-Positive Progenitor Cells in Livestock to Generate a Three-Dimensional Meat Bud

Cited by 13 publications

References 20 publications

Conservation of Markers and Stemness in Adipose Stem and Progenitor Cells between Cattle and Other Species

Conservation of Markers and Stemness in Adipose Stem and Progenitor Cells between Cattle and Other Species

Validation of multiparametric panels for bovine mesenchymal stromal cell phenotyping

Repeated and long-term cryopreservation of primary bovine myogenic cells to maintain quality in biomimetic cultured meat

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