Maintenance of Murine Embryonic Stem Cells' Self-Renewal and Pluripotency with Increase in Proliferation Rate by a Bovine Granulosa Cell Line-Conditioned Medium

Abstract

Murine embryonic stem cells (mESCs) are pluripotent cells that can be propagated in an undifferentiated state in continuous culture on a feeder layer or without feeders in the presence of leukemia inhibitory factor (LIF). Although there has been a great advance since their establishment, ESC culture is still complex and expensive. Therefore, finding culture conditions that maintain the self-renewal of ESCs, preventing their differentiation and promoting their proliferation, is still an area of great interest. In this work, we studied the effects of the conditioned medium from a bovine granulosa cell line (BGC-CM) on the maintenance of self-renewal and pluripotency of mESCs. We found that this medium is able to maintain mESCs' self-renewal while preserving its critical properties without LIF addition. mESCs cultured in BGC-CM expressed the stem cell markers Oct4, Sox2, Nanog, SSEA-1, Klf4, Rex1, and ECAT1. Moreover, mESCs cultured in BGC-CM gave rise to embryoid bodies and teratomas that differentiated effectively to diverse cell populations from endoderm, mesoderm, and ectoderm. Further, we found that mESCs cultured in BGC-CM have an increased proliferation rate compared with cells grown in the mESC standard culture medium supplemented with LIF. These findings may provide a powerful tool to culture mESCs for long periods of time with high proliferation rate while preserving its basic characteristics, contributing to the application of these cells to assess potential tissue engineering and cellular therapy applications.

Manganese Superoxide Dismutase Gene Expression Is Induced by Nanog and Oct4, Essential Pluripotent Stem Cells’ Transcription Factors

“…R1 and Ainv15 ESC lines are commercial lines purchased from ATCC. They were cultured and differentiated as previously described [ 13 – 15 ]. The iPSC-20 line was derived and validated previously by us [ 16 ], and cultured and differentiated as previously described [ 16 ].…”

EDA-Containing Fibronectin Increases Proliferation of Embryonic Stem Cells

“…The E14-derived Ainv15 and R1 mESC lines were obtained from ATCC and cultured as previously described [ 11 , 12 , 20 ].…”

“…We have recently shown that conditioned medium (CM) from a bovine granulosa cell line (BGC-CM) is able to maintain mouse pluripotent stem cells’ self-renewal, including ESC and induced pluripotent stem cells (iPSC), while preserving their unique properties, in culture without Leukemia Inhibitor Factor (LIF) addition [ 11 , 12 ]. Pluripotent stem cells growing on BGC-CM expressed stem cell markers and remained pluripotent.…”

“…Pluripotent stem cells growing on BGC-CM expressed stem cell markers and remained pluripotent. Moreover, we also found that mES cells cultured in these conditions have an increased proliferation rate compared with cells cultured in ESC standard proliferation medium (PM) [ 12 ]. The conditioner cell line was previously established [ 13 ] and formerly selected by its mitogenic properties on the same granulosa cell line and on primary cultures [ 14 ].…”

Tissue inhibitor of metalloproteinase proteins inhibit teratoma growth in mice transplanted with pluripotent stem cells

“…Different types of stem cell‐derived CM have shown various effects on ESCs or iPSCs, such as maintenance of the self‐renewal of ESCs and iPSCs without LIF addition and efficient differentiation of ESCs or iPSCs . Recently, CM has shown therapeutic efficacy in acute lung injury, stroke, and Huntington's disease .…”