Feeder-Free Culture of Murine Induced Pluripotent Stem Cells

Summary

This study describes a method to culture murine induced pluripotent stem cells (iPSCs) independent of feeder cells.

Abstract

Induced pluripotent stem cells (iPSCs), reprogrammed from somatic cells, can self-renew and differentiate into all cell types of the adult body. Typically, murine iPSCs are co-cultured with feeder cells, which supply various undefined growth factors and extracellular matrix components. To ensure experimental results stem exclusively from iPSCs, we aimed to establish a feeder-independent culture system suitable for maintaining murine fibroblast-derived iPSCs. Here, we have investigated culturing murine iPSCs on gelatin-coated plates in the presence of leukemia inhibitory factor (LIF). After five passages, most iPSCs that were previously established and maintained on feeder cells adapted to the feeder-cell-free culture conditions. Morphological characteristics of iPSCs cultured in the presence or absence of feeder cells were similar, with compact colonies and cytoplasmic membrane contact among the cells. Additionally, the absence of feeder cells had no impact on the proliferation of iPSCs. Importantly, the expression of pluripotency and differentiation markers in murine iPSCs was not affected when cultured in the absence of feeder cells after thirty passages. These results suggest that iPSCs cultured without feeder cells maintain their pluripotency. In summary, we have successfully developed a highly efficient system for cultivating murine iPSCs without feeder cells.

Introduction

Induced pluripotent stem cells (iPSCs) are generated by inducing somatic cells into a pluripotent state¹. Similar to embryonic stem cells (ESCs), iPSCs are capable of self-renewal and differentiating to various cell types. Reprogramming a patient's adult cells into iPSCs holds significant promise for studying and potentially treating various diseases by generating patient-specific pluripotent cell lines². In comparison to ESCs, use of iPSCs holds several advantages, including elimination of ethical concerns, mitigation of the immune rejection risk during autologous transplantation, and an unlimited supply of human ce....

Protocol

1. Preparing feeder cells

1. Seed murine MEF C3H/10T1/2 (ATCC, CCL-226, 1.5 x 10⁶) in a 15-cm cell culture dish with medium (20 mL). Culture C3H/10T1/2 cells at 37 ^oC in a 5% CO₂ humidified incubator for 48 h.
   NOTE: C3H/10T1/2 cell culture medium is composed of DMEM, fetal bovine serum (10%), penicillin (50 U/mL), and streptomycin (50 µg/mL).
2. Aspirate the medium of cultured C3H/10T1/2 cells. Rinse the cells with trypsin (5 mL; 0.05%). Incubate the cells with fresh trypsin (5 mL) for 5 min at 37 °C.
3. Resuspend C3H/10T1/2 cells incubated with trypsin. Inactivate trypsin using ....

Results

The morphological characteristics of murine iPSCs cultured in the presence or absence of feeder cells are comparable.
Murine iPSC cell lines TTF-47 and TTF-49 are known to give rise to high-grade chimaeras and support viable "all-iPSC" mice¹³. TTF-47 and TTF-49 cells have been engineered to express green fluorescent protein (GFP) to facilitate the identification of "all-iPSC" mice¹³. We sought to develop a feeder-free culture system for TTF-47 and TTF-4.......

Discussion

Tremendous efforts have been devoted to developing experimental approaches to maintain the proliferation and pluripotency of iPSCs independent of feeder cells¹⁸. Various extracellular matrices have been explored to substitute feeder cells, including Matrigel¹⁹, synthetic polymers¹⁹, and purified proteins²⁰. Gelatin, a degraded form of collagen, is commonly used in feeder-free culture of pluripotent stem cells. Gelatin mimics t.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 647 mouse IgG3, κ isotype control	Becton Dickinson	51-9006270	Immunolabeling (Clone: J606)
Alexa Fluor647 mouse anti-SSEA-4 monoclonal antibody	Becton Dickinson	51-9006265	Immunolabeling (Clone: MC813-70 )
CellQuest Pro Software	Becton Dickinson		Analyzing flowcytometry data
Centrifuge	Eppendorf	5417C	Low speed centrifugation
Centrifuge, Sorvall Legend RT	Thermo Fisher		Low speed centrifugation
Doxycycline monohydrate	Millipore Sigma	D1822	Medium for iPSCs
Dulbeccos Modified Eagles Medium, High Glucose	Cytiva	SH30243.LS	Medium for feeder cells
EVOS M5000  imaging system	Thermo Fisher		Microscopy
FACScalibur flow cytometer	Becton Dickinson		Examining GFP levels of ES-D3 cells
Fetal bovine serum	ATCC	SCRR-30-2020	Medium for iPSCs and feeder cells
Gelatin (0.1%)	Thermo Fisher	ES006B	Culturing iPSCs
Hemacytometer	Hausser Scientific		Measuring cell number
KnockOut Dulbecco’s Modified Eagle’s Medium	Thermo Fisher	10-829-018	Medium for iPSCs
Leukemia Inhibitory Factor	Thermo Fisher	ESG1106	Medium for iPSCs
L-glutamine	VWR	VWRL0131-0100	Medium for iPSCs
Mitomycin C	Cayman	11435	Inhibiting  feeder cell proliferation
Non-essential amino acids	Thermo Fisher	SH3023801	Medium for iPSCs
PE mouse anti-SSEA-1 monoclonal antibody	Becton Dickinson	51-9006268	Immunolabeling (Clone: MC480)
PE mouse IgM, κ isotype control	Becton Dickinson	51-9006273	Immunolabeling (Clone: G155-228)
Penicillin/streptomycin	VWR	sc45000-652	Medium for iPSCs and feeder cells
PerCP-Cy5.5 mouse anti-Oct3/4 monoclonal antibody	Becton Dickinson	51-9006267	Immunolabeling (Clone: 40/Oct-3)
PerCP-Cy5.5 mouse IgG1  κ isotype control	Becton Dickinson	51-9006272	Immunolabeling (Clone: X40)
Prism 10	GraphPad		Statistical analysis
Stemflow Human and Mouse Pluripotent Stem Cell Analysis Kit	Beckon Dickinson	560461	Evaluating pluripotency
Trypan Blue solution	Thermo Fisher	15250061	Counting the number of viable cells
Trypsin	VWR	45000-660	Culturing iPSCs and feeder cells
β-mercaptoethanol	Thermo Fisher	21985023	Medium for iPSCs