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Adult Murine Pancreatic Progenitors Require Epidermal Growth Factor and Nicotinamide for Self-Renewal and Differentiation in a Serum- and Conditioned Medium-Free Culture.

ABSTRACT: Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and tri-lineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture components, such as serum and conditioned medium, has prevented a complete understanding of the signals required for progenitor cell growth. Here, we have established a serum-free, conditioned medium-free colony assay with the inclusion of seven defined factors: epidermal growth factor (EGF), R-Spondin 1 (RSPO1), Noggin, nicotinamide, exendin-4, activin B, and vascular endothelial growth factor (VEGF)-A. The requirements for colony growth were characterized and we found that EGF and nicotinamide were necessary and sufficient for the colony growth and long-term self-renewal of these progenitors. However, the seven factor (7F) culture medium better induced colony size and self-renewal in long-term culture than EGF plus nicotinamide alone. Individual 3-week-old colonies grown in the 7F culture medium expressed ductal, acinar, and endocrine lineage markers, suggesting that tri-lineage differentiation of the tri-potent progenitors was occurring without genetic manipulation. A delayed inhibition of Notch signaling using small molecules in 2-week-old cultures enhanced endocrine gene expression in 3-week-old colonies. This better-defined colony assay system will enable our and other laboratories for in-depth mechanistic studies on the biology of these progenitor cells.

SUBMITTER: Wedeken L

PROVIDER: S-EPMC5393420 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Adult Murine Pancreatic Progenitors Require Epidermal Growth Factor and Nicotinamide for Self-Renewal and Differentiation in a Serum- and Conditioned Medium-Free Culture.

Wedeken Lena L Luo Angela A Tremblay Jacob R JR Rawson Jeffrey J Jin Liang L Gao Dan D Quijano Janine J Ku Hsun Teresa HT

Stem cells and development 20170117 8

Adult pancreatic stem and progenitor cells may serve as an alternative source of insulin-secreting endocrine cells in cell replacement therapy for type 1 diabetes, but much remained unknown about these cells. We previously identified adult murine pancreatic progenitor-like cells that displayed in vitro self-renewal and tri-lineage differentiation activities in a three-dimensional colony/organoid assay containing 1% methylcellulose and 5% Matrigel. However, the presence of other undefined culture ...[more]

PMID: 28095743

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Project description:BACKGROUND: Notochordal cells (NC) remain in the focus of research for regenerative therapy for the degenerated intervertebral disc (IVD) due to their progenitor status. Recent findings suggested their regenerative action on more mature disc cells, presumably by the secretion of specific factors, which has been described as notochordal cell conditioned medium (NCCM). The aim of this study was to determine NC culture conditions (2D/3D, fetal calf serum, oxygen level) that lead to significant IVD cell activation in an indirect co-culture system under normoxia and hypoxia (2% oxygen). METHODS: Porcine NC was kept in 2D monolayer and in 3D alginate bead culture to identify a suitable culture system for these cells. To test stimulating effects of NC, co-cultures of NC and bovine derived coccygeal IVD cells were conducted in a 1:1 ratio with no direct cell contact between NC and bovine nucleus pulposus cell (NPC) or annulus fibrosus cells (AFC) in 3D alginate beads under normoxia and hypoxia (2%) for 7 and 14 days. As a positive control, NPC and AFC were stimulated with NC-derived conditioned medium (NCCM). Cell activity, glycosaminoglycan (GAG) content, DNA content and relative gene expression was measured. Mass spectrometry analysis of the NCCM was conducted. RESULTS: We provide evidence by flow cytometry that monolayer culture is not favorable for NC culture with respect to maintaining NC phenotype. In 3D alginate culture, NC activated NPC either in indirect co-culture or by addition of NCCM as indicated by the gene expression ratio of aggrecan/collagen type 2. This effect was strongest with 10% fetal calf serum and under hypoxia. Conversely, AFC seemed unresponsive to co-culture with pNC or to the NCCM. Further, the results showed that hypoxia led to decelerated metabolic activity, but did not lead to a significant change in the GAG/DNA ratio. Mass spectrometry identified connective tissue growth factor (CTGF, syn. CCN2) in the NCCM. CONCLUSIONS: Our results confirm the requirement to culture NC in 3D to best maintain their phenotype, preferentially in hypoxia and with the supplementation of FCS in the culture media. Despite these advancements, the ideal culture condition remains to be identified.

Project description:This study aimed to assess the impact of conditioned medium from epidermal neural crest stem cells (EPI-NCSCs-CM) on functional recovery following spinal cord injury (SCI), while also exploring the involvement of the PI3K-AKT signaling pathway in regulating neuronal apoptosis. EPI-NCSCs were isolated from 10-day-old Sprague-Dawley rats and cultured for 48 h to obtain EPI-NCSC-CM. SHSY-5Y cells were subjected with H2O2 treatment to induce apoptosis. Cell viability and survival rates were evaluated using the CCK-8 assay and calcein-AM/PI staining. SCI contusion model was established in adult Sprague-Dawley rats to assess functional recovery, utilizing the Basso, Beattie and Bresnahan (BBB) scoring system, inclined test, and footprint observation. Neurological restoration after SCI was analyzed through electrophysiological recordings. Histological analysis included hematoxylin and eosin (H&E) staining and Nissl staining to evaluate tissue organization. Apoptosis and oxidative stress levels were assessed using TUNEL staining and ROS detection methods. Additionally, western blotting was performed to examine the expression of apoptotic markers and proteins related to the PI3K/AKT signaling pathway. EPI-NCSC-CM significantly facilitated functional and histological recovery in SCI rats by inhibiting neuronal apoptosis through modulation of the PI3K/AKT pathway. Administration of EPI-NCSCs-CM alleviated H2O2-induced neurotoxicity in SHSY-5Y cells in vitro. The use of LY294002, a PI3K inhibitor, underscored the crucial role of the PI3K/AKT signaling pathway in regulating neuronal apoptosis. This study contributes to the ongoing exploration of molecular pathways involved in spinal cord injury (SCI) repair, focusing on the therapeutic potential of EPI-NCSC-CM. The research findings indicate that EPI-NCSC-CM exerts a neuroprotective effect by suppressing neuronal apoptosis through activation of the PI3K/AKT pathway in SCI rats. These results highlight the promising role of EPI-NCSC-CM as a potential treatment strategy for SCI, emphasizing the significance of the PI3K/AKT pathway in mediating its beneficial effects.

Dataset Information

Adult Murine Pancreatic Progenitors Require Epidermal Growth Factor and Nicotinamide for Self-Renewal and Differentiation in a Serum- and Conditioned Medium-Free Culture.

Publications

Adult Murine Pancreatic Progenitors Require Epidermal Growth Factor and Nicotinamide for Self-Renewal and Differentiation in a Serum- and Conditioned Medium-Free Culture.

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