Porcine Primordial Germ Cell-Like Cells Generated from Induced Pluripotent Stem Cells Under Different Culture Conditions

Pieri, Naira Caroline Godoy; Souza, Aline Fernanda de; Botigelli, Ramon César; Pessôa, Laís Vicari de Figueirêdo; Recchia, Kaiana; Machado, Liliane S.; Glória, Mayra Hirakawa; Castro, Raquel Vasconcelos Guimarães de; Leal, Diego Feitosa; Neto, Paulo Fantinato; Martins, Simone Maria Massami Kitamura; Martins, Daniele dos Santos; Bressan, Fabiana Fernandes; Andrade, André Furugen Cesar de

doi:10.1007/s12015-021-10198-8

Cited by 19 publications

(33 citation statements)

References 86 publications

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“…In addition, no morphological differences during the reprogramming process were observed between the different medium supplementations in this study. Whereas the exact mechanisms behind the different media requirements necessary to maintain the pluripotency of iPSCs generated from different cell lines have not yet been elucidated in large animal models, we have experienced similar outcomes in previous studies of other species [ 12 , 15 , 18 ].…”

Section: Discussionsupporting

confidence: 63%

Influence of Cell Type in In Vitro Induced Reprogramming in Cattle

Recchia

Pessôa

Pieri

et al. 2022

Life

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Induced pluripotent stem cells (iPSCs) have been considered an essential tool in stem cell research due to their potential to develop new therapies and technologies and answer essential questions about mammalian early development. An important step in generating iPSCs is selecting their precursor cell type, influencing the reprogramming efficiency and maintenance in culture. In this study, we aim to characterize bovine mesenchymal cells from adipose tissue (bAdMSCs) and fetal fibroblasts (bFFs) and to compare the reprogramming efficiency of these cells when induced to pluripotency. The cells were characterized by immunostaining (CD90, SSEA1, SSEA3, and SSEA4), induced differentiation in vitro, proliferation rates, and were subjected to cell reprogramming using the murine OSKM transcription factors. The bFFs presented morphological changes resembling pluripotent cells after reprogramming and culture with different supplementation, and putative iPSCs were characterized by immunostaining (OCT4, SOX2, NANOG, and AP). In the present study, we demonstrated that cell line origin and cellular proliferation rate are determining factors for reprogramming cells into pluripotency. The generation of biPSCs is a valuable tool to improve both translational medicine and animal production and to study the different supplements required to maintain the pluripotency of bovine cells in vitro.

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Section: Discussionsupporting

confidence: 63%

Influence of Cell Type in In Vitro Induced Reprogramming in Cattle

Recchia

Pessôa

Pieri

et al. 2022

Life

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“…Primordial germ-cells-like cells (PGCLCs) were generated in vitro from both iPSCs and ESCs, even generating viable offspring in mice [259]. This methodology is a significant advancement in the field of assisted reproduction and animal preservation [162], and has been applied to different species, such as mice [259][260][261], humans [262,263], nonhuman primates [264], pigs [265], and goats [266], although more robust results were achieved in mice. Supplementation with FSH, bovine pituitary extract, and testosterone resulted in an increased percentage of mouse ES-derived PGCLCS completing meiosis in vitro, resulting in spermatid-like cells (SLCs) [267].…”

Section: Influence Of Fsh In Germ Cells In Vitromentioning

confidence: 99%

Actions and Roles of FSH in Germinative Cells

Recchia

Jorge

Pessôa

et al. 2021

IJMS

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Follicle stimulating hormone (FSH) is produced by the pituitary gland in a coordinated hypothalamic–pituitary–gonadal (HPG) axis event, plays important roles in reproduction and germ cell development during different phases of reproductive development (fetal, neonatal, puberty, and adult life), and is consequently essential for fertility. FSH is a heterodimeric glycoprotein hormone of two dissociable subunits, α and β. The FSH β-subunit (FSHβ) function starts upon coupling to its specific receptor: follicle-stimulating hormone receptor (FSHR). FSHRs are localized mainly on the surface of target cells on the testis and ovary (granulosa and Sertoli cells) and have recently been found in testicular stem cells and extra-gonadal tissue. Several reproduction disorders are associated with absent or low FSH secretion, with mutation of the FSH β-subunit or the FSH receptor, and/or its signaling pathways. However, the influence of FSH on germ cells is still poorly understood; some studies have suggested that this hormone also plays a determinant role in the self-renewal of germinative cells and acts to increase undifferentiated spermatogonia proliferation. In addition, in vitro, together with other factors, it assists the process of differentiation of primordial germ cells (PGCLCs) into gametes (oocyte-like and SSCLCs). In this review, we describe relevant research on the influence of FSH on spermatogenesis and folliculogenesis, mainly in the germ cell of humans and other species. The possible roles of FSH in germ cell generation in vitro are also presented.

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“…Indeed, the same human/mouse protocols for iPSCs generation are not extendable to other species[ 1 , 3 ]. Herein we used a previous strategy already reported for large animals reprogramming[ 34 - 36 , 43 - 45 ], and widely used to reprogram porcine somatic cells[ 1 ]. In the conditions described, the results showed that UDCs transduced with human factors failed to be maintained in culture for more than 5 passages due to early differentiation of the cells cultured in vitro , and similar results was described by Pieri et al [ 36 ] when reprogramming of porcine foetal fibroblasts.…”

Section: Discussionmentioning

confidence: 99%

“…Herein we used a previous strategy already reported for large animals reprogramming[ 34 - 36 , 43 - 45 ], and widely used to reprogram porcine somatic cells[ 1 ]. In the conditions described, the results showed that UDCs transduced with human factors failed to be maintained in culture for more than 5 passages due to early differentiation of the cells cultured in vitro , and similar results was described by Pieri et al [ 36 ] when reprogramming of porcine foetal fibroblasts. Conversely, cells transduced with murine factors were maintained in culture for at least 28 passages, showing typical morphology, positive AP detection and endogenous pluripotency-related gene expression through the different passages.…”

Section: Discussionmentioning

confidence: 99%

“…The cultured putative piPSCs were fixed in paraformaldehyde for 10 min and washed in PBS. The pluripotency-related markers test was performed as previously described[ 36 ]. Briefly, the antibodies were used to detect OCT4 (1:100, cat# SC8628, Santa Cruz), SOX2 (1:500, cat# ab97959; Abcam), NANOG (1:100, cat# ab77095, Abcam), SSEA1 (1:50, cat# MAB4301, Millipore) and TRA1 81 (1:50, cat# MAB4381, Millipore), and the respective secondary antibodies were used (donkey anti-goat 594, cat# A11058, donkey anti-rabbit 488, A21206, 1:500, donkey anti-goat 488, cat# A11055, Invitrogen, 1:500 goat anti-mouse 594, cat# A21044, Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

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In vitro induced pluripotency from urine-derived cells in porcine

Recchia¹,

Machado²,

Botigelli³

et al. 2022

WJSC

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BACKGROUND The generation of induced pluripotent stem cells (iPSC) has been a game-changer in translational and regenerative medicine; however, their large-scale applicability is still hampered by the scarcity of accessible, safe, and reproducible protocols. The porcine model is a large biomedical model that enables translational applications, including gene editing, long term in vivo and offspring analysis; therefore, suitable for both medicine and animal production. AIM To reprogramme in vitro into pluripotency, and herein urine-derived cells (UDCs) were isolated from porcine urine. METHODS The UDCs were reprogrammed in vitro using human or murine octamer-binding transcription factor 4 (OCT4), SRY-box2 (SOX2), Kruppel-like factor 4 (KLF4), and C-MYC, and cultured with basic fibroblast growth factor (bFGF) supplementation. To characterize the putative porcine iPSCs three clonal lineages were submitted to immunocytochemistry for alkaline phosphatase (AP), OCT4, SOX2, NANOG, TRA1 81 and SSEA 1 detection. Endogenous transcripts related to the pluripotency (OCT4, SOX2 and NANOG) were analyzed via reverse transcription quantitative real-time polymerase chain reaction in different time points during the culture, and all three lineages formed embryoid bodies (EBs) when cultured in suspension without bFGF supplementation. RESULTS The UDCs were isolated from swine urine samples and when at passage 2 submitted to in vitro reprogramming. Colonies of putative iPSCs were obtained only from UDCs transduced with the murine factors (mOSKM), but not from human factors (hOSKM). Three clonal lineages were isolated and further cultured for at least 28 passages, all the lineages were positive for AP detection, the OCT4, SOX2, NANOG markers, albeit the immunocytochemical analysis also revealed heterogeneous phenotypic profiles among lineages and passages for NANOG and SSEA1, similar results were observed in the abundance of the endogenous transcripts related to pluripotent state. All the clonal lineages when cultured in suspension without bFGF were able to form EBs expressing ectoderm and mesoderm layers transcripts. CONCLUSION For the first time UDCs were isolated in the swine model and reprogrammed into a pluripotent-like state, enabling new numerous applications in both human or veterinary regenerative medicine.

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Porcine Primordial Germ Cell-Like Cells Generated from Induced Pluripotent Stem Cells Under Different Culture Conditions

Cited by 19 publications

References 86 publications

Influence of Cell Type in In Vitro Induced Reprogramming in Cattle

Influence of Cell Type in In Vitro Induced Reprogramming in Cattle

Actions and Roles of FSH in Germinative Cells

In vitro induced pluripotency from urine-derived cells in porcine

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