Choi, Kwang-Hwan
(Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University)
,
Lee, Dong-Kyung
(Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University)
,
Oh, Jong-Nam
(Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University)
,
Kim, Seung-Hun
(Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University)
,
Lee, Mingyun
(Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University)
,
Jeong, Jinsol
(Department of Agricultural Biotechnology, Animal Biotechnology Major, and Research Institute of Agriculture and Life Science, Seoul National University)
,
Choe, Gyung Cheol
(Department of Agricultural Biotechnology, Animal Biotechnology Major, and Re)
,
Lee, Chang-Kyu
As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neurona...
As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.
As a preclinical study, many researchers have been attempted to convert the porcine PSCs into several differentiated cells with transplantation of the differentiated cells into the pigs. Here, we attempted to derive neuronal progenitor cells from pig embryonic germ cells (EGCs). As a result, neuronal progenitor cells could be derived directly from pig embryonic germ cells through the serum-free floating culture of EB-like aggregates (SFEB) method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs rather than inhibiting SMAD signaling. The differentiated cells expressed neuronal markers such as PAX6, NESTIN, and SOX1 as determined by qRT-PCR and immunostaining. These data indicated that pig EGCs could provide valid models for human therapy. Finally, it is suggested that developing transgenic pig for disease models as well as differentiation methods will provide basic preclinical data for human regenerative medicine and lead to the success of stem cell therapy.
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문제 정의
To apply pig PSCs for a therapeutic model, a development of methods for directed differentiation into specific lineages are important. For this reason, this study aimed to develop a method for inducing neural lineage from pig EGCs. It has been verified that several signaling including SMAD inhibition and retinoic acid are involved in differentiation into neural cells from pluripotent cells (Parsons et al.
가설 설정
To find which molecules are most efficient in inducing neural cells from pig EGCs, SMAD signaling inhibitors and retinoic acid were selected. (B) Embryoid bodies were formed by suspension culture. (C) After plating onto matrigel, they were cultured for 8 days.
제안 방법
Amplification and detection were conducted using the ABI 7300 Real-Time PCR system (Applied Biosystems) under the following conditions: one cycle of 50℃ for 2 min and 95℃ for 10 min, followed by 40 cycles of denaturation at 95℃ for 15 sec and annealing/extension for 1 min (annealing/extension temperatures depended on each primer set).
Cultured EGCs were dissociated into single cells using 0.25% trypsin/EDTA solution (Welgene) and cultured in Ultra-Low attachment plates (Sigma Aldrich, MO, USA) with STEMdiffTM Neural Induction Medium (STEMCELL, Vancouver, Canada) containing 5 µM retinoic acid (RA) and SMAD signaling inhibitors (SMADi; 400 µM Noggin and 2 µM SB431542) for 5 days.
(A) Differentiation of neural lineage was accomplished according to the serum-free floating culture of EB-like aggregates (SFEB) methods with some modifications. Differentiation was conducted with the porcine embryonic stem cell media (PESM) as a control media and the neural induction media (NIM). To find which molecules are most efficient in inducing neural cells from pig EGCs, SMAD signaling inhibitors and retinoic acid were selected.
데이터처리
Statistical differences between datasets were determined by one-way analyses of variance (ANOVAs) followed by Fisher’s least significant difference (LSD) tests.
이론/모형
Based on our results, neuronal progenitor cells could be derived directly from pig EGCs through the SFEB method. Treating retinoic acid was more efficient for inducing neuronal lineages from EGCs than inhibiting SMAD signaling.
In this study, we attempted to derive neuronal progenitor cells from pig EGCs according to the serum-free floating culture of the EB-aggregates (SFEB) method (Watanabe et al., 2005). Similar to other species, neuronal progenitor cells were successfully induced by treatment of retinoic acid and these cells expressed neuronal markers such as PAX6, NESTIN, and SOX1.
참고문헌 (29)
Ao Y, Mich-Basso JD, Lin B, Yang L. 2014. High efficient differentiation of functional hepatocytes from porcine induced pluripotent stem cells. PLoS One 9:e100417.
Choi KH, Lee DK, Kim SW, Woo SH, Kim DY, Lee CK. 2019. Chemically defined media can maintain pig pluripotency network in vitro. Stem Cell Reports 13:221-234.
Choi KH, Lee DK, Oh JN, Son HY, Lee CK. 2018. FGF2 signaling plays an important role in maintaining pluripotent state of pig embryonic germ cells. Cell. Reprogram. 20:301-311.
Eiraku M, Watanabe K, Matsuo-Takasaki M, Kawada M, Yonemura S, Matsumura M, Wataya T, Nishiyama A, Muguruma K, Sasai Y. 2008. Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. Cell Stem Cell 3:519-532.
Espuny-Camacho I, Michelsen KA, Gall D, Linaro D, Hasche A, Bonnefont J, Bali C, Orduz D, Bilheu A, Herpoel A, Lambert N, Gaspard N, Peron S, Schiffmann SN, Giugliano M, Gaillard A, Vanderhaeghen P. 2013. Pyramidal neurons derived from human pluripotent stem cells integrate efficiently into mouse brain circuits in vivo. Neuron 77:440-456.
Gallegos-Cardenas A, Webb R, Jordan E, West R, West FD, Yang JY, Wang K, Stice SL. 2015. Pig induced pluripotent stem cell-derived neural rosettes developmentally mimic human pluripotent stem cell neural differentiation. Stem Cells Dev. 24:1901-1911.
Kragh PM, Nielsen AL, Li J, Du Y, Lin L, Schmidt M, Bogh IB, Holm IE, Jakobsen JE, Johansen MG, Purup S, Bolund L, Vajta G, Jorgensen AL. 2009. Hemizygous minipigs produced by random gene insertion and handmade cloning express the Alzheimer's disease-causing dominant mutation APPsw. Transgenic Res. 18:545-558.
Lancaster MA, Renner M, Martin CA, Wenzel D, Bicknell LS, Hurles ME, Homfray T, Penninger JM, Jackson AP, Knoblich JA. 2013. Cerebral organoids model human brain development and microcephaly. Nature 501:373-379.
Li X, Zhang F, Song G, Gu W, Chen M, Yang B, Li D, Wang D, Cao K. 2013. Intramyocardial injection of pig pluripotent stem cells improves left ventricular function and perfusion: a study in a porcine model of acute myocardial infarction. PLoS One 8:e66688.
Livak KJ and Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402-408.
Lorson MA, Spate LD, Samuel MS, Murphy CN, Lorson CL, Prather RS, Wells KD. 2011. Disruption of the Survival Motor Neuron (SMN) gene in pigs using ssDNA. Transgenic. Res. 20:1293-1304.
Park KM, Hussein KH, Ghim JH, Ahn C, Cha SH, Lee GS, Hong SH, Yang S, Woo HM. 2015. Hepatic differentiation of porcine embryonic stem cells for translational research of hepatocyte transplantation. Transplant. Proc. 47:775-779.
Parsons XH, Teng YD, Parsons JF, Snyder EY, Smotrich DB, Moore DA. 2011. Efficient derivation of human neuronal progenitors and neurons from pluripotent human embryonic stem cells with small molecule induction. J. Vis. Exp. (56):e3273.
Puy Ld, Chuva de Sousa Lopes SM, Haagsman HP, Roelen BA. 2010. Differentiation of porcine inner cell mass cells into proliferating neural cells. Stem Cells Dev. 19:61-70.
Suga H, Kadoshima T, Minaguchi M, Ohgushi M, Soen M, Nakano T, Takata N, Wataya T, Muguruma K, Miyoshi H, Yonemura S, Oiso Y, Sasai Y. 2011. Self-formation of functional adenohypophysis in three-dimensional culture. Nature 480:57-62.
Wang H, Xiang J, Zhang W, Li J, Wei Q, Zhong L, Ouyang H, Han J. 2016. Induction of germ cell-like cells from porcine induced pluripotent stem cells. Sci. Rep. 6:27256.
Zhang F, Song G, Li X, Gu W, Shen Y, Chen M, Yang B, Qian L, Cao K. 2014. Transplantation of iPSc ameliorates neural remodeling and reduces ventricular arrhythmias in a postinfarcted swine model. J. Cell. Biochem. 115:531-539.
Zhou L, Wang W, Liu Y, Fernandez de Castro J, Ezashi T, Telugu BP, Roberts RM, Kaplan HJ, Dean DC. 2011. Differentiation of induced pluripotent stem cells of swine into rod photoreceptors and their integration into the retina. Stem Cells 29:972-980.
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