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 cell types needed for therapeutic treatments^3,4.

Murine iPSCs can be generated by ectopically overexpressing a defined set of transcription factors, including Oct3/4, Sox2, c-Myc, and Klf4 in somatic cells^5,6. Studies have shown that genetically identical murine ESCs and iPSCs exhibit no differences in biological function, transcription profiles, or epigenetic states⁷. Similar to ESCs, iPSCs produce various types of differentiated cells. More importantly, murine iPSCs display an embryonic level of pluripotent potential by generating full-term, live progeny from complemented tetraploid blastocysts^8,9.

Murine iPSCs are typically cultured with feeder cells, usually composed of non-dividing mouse embryonic fibroblasts (MEFs), which create a supportive environment for maintaining pluripotency¹⁰. Published work indicates that feeder cells function by supplying iPSCs with undefined growth factors and extracellular matrix components¹¹. To achieve cell cycle arrest, feeder cells are mitotically inactivated by either irradiation or by the use of a potent DNA crosslinker, such as mitomycin C¹². However, co-culturing iPSCs with feeder cells can be challenging, further complicated by the unknown nature of interactions between them¹⁰. Moreover, the presence of feeder cells makes it difficult to accurately evaluate biological functions of iPSCs and limits the clinical applications of iPSCs. Therefore, it is imperative to develop feeder-cell-free systems for culturing iPSCs.

In this study, we studied two murine iPSC cell lines derived from tail-tip fibroblasts (TTF), TTF-47 and TTF-49. These lines have demonstrated the ability to contribute to high-grade chimeras and support the development of postnatal "all-iPSC" mice¹³. We have described a detailed method to efficiently culture murine iPSC lines TTF-47 and TTF-49 without feeder cells. We have compared the morphology, proliferation, and pluripotent marker expression of these cells grown under different culture conditions. In conclusion, feeder-free cultured iPSCs show great potential as valuable tools for exploring their therapeutic applications.

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 C3H/10T1/2 culture medium (5 mL).
4. Collect the cells by centrifugation (390 g) for 5 min. Disperse the cells into a single-cell suspension using C3H/10T1/2 culture medium (2 mL).
5. Mix the medium containing C3H/10T1/2 cells and trypan blue solution (0.4%) at 1:1 ratio. Determine the number of viable cells using a hemocytometer.
6. Adjust the concentration of C3H/10T1/2 cells to 2 × 10⁶/mL. Add mitomycin C at a final concentration of 25 µg/mL.
7. Incubate the cells at 37 °C cell culture incubator for 1 h. Mix cells every 10-15 min with gentle agitation.
8. Centrifuge C3H/10T1/2 cells at 390 g for 5 min. Remove the medium. Wash the cells twice with fresh medium (10 mL).
9. Resuspend the cells in fresh medium (5 mL). Determine the number of viable cells using a hemocytometer.
10. Plate 8 × 10⁵ C3H/10T1/2 cells in a 10-cm cell culture dish (1.4 × 10⁴ cells per cm²) with medium (10 mL). Culture the cells in a cell culture incubator at 37 °C for 24 h.

2. Culturing iPSCs in the presence of feeder cells

1. Thawing frozen iPSCs
   1. Thaw a vial of frozen iPSC line TTF-47 or TTF-49 (5 × 10⁶ cells; received from Dr. Hochedlinger; Massachusetts General Hospital, Boston, MA) in a 37 °C water bath for 1 min with gentle agitation.
   2. Transfer TTF-47 or TTF-49 cells into a 15-mL conical tube. Add pre-warmed iPSC medium (10 mL) gradually to prevent damage to the cells caused by osmotic changes.
      NOTE: iPSC medium for TTF-47 and TTF-49 cells is composed of DMEM, fetal bovine serum (15%), non-essential amino acids (0.1 mM), L-glutamine (2 mM), β-mercaptoethanol (0.1 mM), penicillin (50 U/mL), streptomycin (50 µg/mL), doxycycline (1 µg/mL), and leukemia inhibitory factor (LIF; 100 units/mL).
   3. Centrifuge at 390 g for 5 min at room temperature (RT). Aspirate the medium. Resuspend TTF-47 or TTF-49 cells in iPSC medium (10 mL).
   4. Aspirate medium of a 10-cm dish pre-seeded with mitomycin-C-treated C3H/10T1/2 cells (8 × 10⁵) described in step 1.10. Transfer TTF-47 or TTF-49 cells in iPSC medium to the dish with mitomycin-C-treated C3H/10T1/2 cells.
   5. Culture the cells in a 37 °C cell culture incubator. Replace the old medium with fresh medium every 3 days.
2. Collecting iPSCs
   1. Aspirate culture medium when the confluency of TTF-47 or TTF-49 cells is 90%. Rinse the cells with trypsin (2 mL). Incubate the cells with fresh trypsin (2 mL) for 5 min at 37 °C.
   2. Resuspend iPSCs and feeder cells treated with trypsin. Inactivate trypsin using iPSC culture medium (2 mL). Collect the cells by centrifugation (390 g) for 5 min.
   3. Disperse the cells into a single-cell suspension using iPSC culture medium (5 mL). Count the number of viable cells with a hemocytometer.
   4. Transfer the medium containing both iPSCs and feeder cells to an uncoated 10-cm cell culture dish. Incubate in a 37 °C cell culture incubator for 30 min so that C3H/10T1/2 cells but not TTF-47 or TTF-49 cells would attach to the bottom of dish.
   5. Collect the medium containing TTF-47 or TTF-49 cells. Determine the number of viable cells using a hemocytometer.
      NOTE: The difference in the numbers of viable cells before and after incubation in an uncoated 10-cm dish should be about the same as the number of feeder cells.

3. Culturing iPSCs in the absence of feeder cells

1. Coat 10-cm cell culture dishes with gelatin (5 mL; 0.1%) at RT for 30 min. Remove gelatin by aspiration.
2. Plate TTF-47 or TTF-49 cells (5 × 10⁵) acquired from a co-culture with C3H/10T1/2 cells described in step 2.2.5 onto a gelatin-coated 10-cm cell culture dish with fresh iPSC medium whose composition is described in the step 2.1 (10 mL). Culture for 3 days without changing medium before sub-culturing the cells.
3. Aspirate the medium of TTF-47 or TTF-49 cells cultured for 3 days. Rinse the cells using trypsin (2 mL). Incubate the cells with fresh trypsin (2 mL) for 5 min at 37 °C.
4. Resuspend trypsin-treated TTF-47 or TTF-49 cells. Inactivate trypsin using iPSC culture medium (2 mL).
5. Collect the cells by centrifugation (390 g) for 5 min. Disperse the cells using fresh iPSC culture medium (5 mL).
6. Determine the number of viable cells using a hemocytometer. Repeat sub-culturing 4 more times. Replace the old medium with fresh medium every 3 days.
   NOTE: After five rounds of sub-culturing, iPSCs adapted to the culture conditions in the absence of feeder cells.

4. Examining the morphology of iPSCs by microscopy

1. Assessing the morphology of iPSCs cultured with feeder cells
   1. Seed mitomycin-C-treated C3H/10T1/2 cells (5.5 × 10⁴) described in step 1.9 to one well of a 12-well cell culture plate in C3H/10T1/2 culture medium (2 mL). Culture the cells in a 37 °C cell culture incubator for 24 h.
   2. Collect TTF-47 or TTF-49 cells co-cultured with mitomycin-C-treated C3H/10T1/2 cells from a 10-cm cell culture dish described in step 2.2.5. Add TTF-47 or TTF-49 cells (5 × 10³) to one well of 12-well cell culture plate in iPSC medium (2 mL). Culture the cells in a 37 °C cell culture incubator for 48 h.
   3. Capture the transmitted light and green fluorescent protein (GFP) fluorescence images of cells using a cell imaging system with a 10x magnification objective lens (numerical aperture [NA]: 0.25).
2. Assessing the morphology of iPSCs cultured without feeder cells
   1. Coat a 12-well cell culture plate using gelatin (0.5 mL; 0.1%) at RT for 30 min. Remove gelatin by aspiration.
   2. Collect TTF-47 or TTF-49 cells cultured without feeder cells from a 10-cm cell culture dish described in step 3.6. Add TTF-47 or TTF-49 cells (5 × 10³) to one well of the 12-well cell culture plate coated with gelatin in iPSC medium (2 mL). Culture the cells in a 37 °C cell culture incubator for 48 h.
   3. Capture the transmitted light and GFP fluorescence images of cells using a cell imaging system with a 10x magnification objective lens (NA: 0.25).

5. Measuring the proliferation of iPSCs

1. Determining the proliferation of iPSCs cultured with feeder cells
   1. Seed mitomycin-C-treated C3H/10T1/2 cells (5.5 × 10⁴) described in step 1.9 to each well of two 12-well cell culture plates in C3H/10T1/2 culture medium (2 mL). Culture the cells in a 37 °C cell culture incubator for 24 h. Replace medium with iPSC medium (2 mL).
   2. Collect TTF-47 or TTF-49 cells co-cultured with mitomycin-C-treated C3H/10T1/2 cells from a 10-cm cell culture dish described in step 2.2.5. Add TTF-47 or TTF-49 cells (5 × 10³) to each well in one 12-well plate containing feeder cells in iPSC medium (2 mL). Culture the cells in a 37 °C cell culture incubator.
   3. After 24 h of culture, aspirate and remove the medium from three wells in the 12-well cell culture plate containing co-cultured TTF-47 or TTF-49 cells with C3H/10T1/2 feeder cells, as well as from the 12-well plate containing only C3H/10T1/2 feeder cells.
   4. Rinse the wells using trypsin (0.5 mL). Incubate the cells with fresh trypsin (0.5 mL) for 5 min at 37 °C.
   5. Resuspend trypsin-treated cells. Inactivate trypsin using iPSC culture medium (0.5 mL). Collect the cells by centrifugation (390 g) for 5 min. Disperse the cells into a single cell suspension using their culture medium (0.1 mL).
   6. Count the number of viable cells with a hemocytometer. Acquire the mean value of the measurements from three wells. Determine the number of TTF-47 or TTF-49 cells by subtracting the value of the wells containing only C3H/10T1/2 cells from the value of the wells containing both iPSCs and feeder cells.
   7. Repeat determining the number of TTF-47 or TTF-49 cells at 48 h, 72 h, and 96 h time-points post-cell seeding.
2. Determining the proliferation of TTF-47 or TTF-49 cells cultured in the absence of feeder cells
   1. Coat a 12-well cell culture plate using gelatin (0.5 mL; 0.1%) at RT for 30 min. Remove gelatin by aspiration.
   2. Collect TTF-47 or TTF-49 cells cultured without feeder cells from a 10-cm cell culture dish described in step 3.6. Seed TTF-47 or TTF-49 cells (5 × 10³) in each well of one 12-well cell culture plate in iPSC medium (2 mL).
   3. After 24 h of culture, aspirate and remove the medium from three wells of the 12-well cell culture plate containing TTF-47 or TTF-49 cells. Rinse the cells using trypsin (0.5 mL).
   4. Incubate the cells with trypsin (0.5 mL) for 5 min at 37 °C. Resuspend the cells treated with trypsin. Inactivate trypsin using iPSC culture medium (0.5 mL).
   5. Collect the cells by centrifugation (390 g) for 5 min. Disperse the cells using iPSC culture medium (0.1 mL).
   6. Use a hemocytometer to determine the number of viable cells. Calculate the average of the measurements from the three wells.
   7. Repeat determining the number of TTF-47 or TTF-49 cells at 48 h, 72 h, and 96 h time-points post-cell seeding.

6. Evaluating the expression of pluripotent and differentiation markers

1. Preparing iPSCs cultured with feeder cells
   1. Seed mitomycin-C-treated C3H/10T1/2 cells (2 × 10⁶) described in step 1.9 in a 15-cm cell culture dish using C3H/10T1/2 culture medium (20 mL). Culture the cells in a 37 °C cell culture incubator for 24 h.
   2. Remove C3H/10T1/2 culture medium by aspiration. Transfer TTF-47 or TTF-49 cells (2 × 10⁶) described in Step 2.2.5 to the dish in iPSC medium (20 mL). Culture the cells in a 37 °C cell culture incubator for 48 h.
   3. Aspirate the medium of cultured iPSCs and feeder cells. Rinse the cells with trypsin (5 mL). Incubate the cells with fresh trypsin (5 mL) for 5 min at 37 °C.
   4. Resuspend iPSCs and feeder cells incubated with trypsin. Inactivate trypsin using iPSC culture medium (5 mL). Collect iPSCs and feeder cells by centrifugation (390 g) for 5 min.
   5. Disperse the cells using iPSC culture medium (15 mL). Count the number of viable cells with a hemocytometer.
   6. Add cells to an uncoated 15-cm cell culture dish. Incubate in a 37 °C cell culture incubator for 30 min to remove feeder cells.
   7. Collect medium containing TTF-47 or TTF-49 cells. Determine the number of viable cells using a hemocytometer.
2. Preparing iPSCs cultured without feeder cells
   1. Seed TTF-47 or TTF-49 cells (2 × 10⁶) described in step 3.6 in a 15-cm cell culture dish coated with gelatin (0.1%) using iPSC medium (20 mL). Culture the cells in a 37 °C cell culture incubator for 48 h.
   2. Aspirate the medium of cultured TTF-47 or TTF-49 cells. Rinse the cells with trypsin (5 mL). Incubate the cells with fresh trypsin (5 mL) for 5 min at 37 °C.
   3. Resuspend trypsin-treated TTF-47 or TTF-49 cells. Inactivate trypsin using iPSC culture medium (5 mL). Collect the cells by centrifugation (390 g) for 5 min.
   4. Disperse the cells using their culture medium (15 mL). Measure the number of viable cells with a hemocytometer.
3. Examining the expression of the pluripotency markers
   1. Label TTF-47 or TTF-49 cells with antibodies specific for SSEA-1, Oct-3/4, and SSEA-4 in an assay kit designed to evaluate the pluripotency of murine pluripotent stem cells following the manufacturer's protocol.
   2. Mix 1 × 10⁶ TTF-47 or TTF-49 cells in 100 μL of 1x PBS with 20 μL of antibodies. Incubate at RT in the dark for 30 min.
   3. Evaluate the expression levels of SSEA-1, Oct-3/4, and SSEA-4 using a flow cytometer. Acquire 10,000 events for each sample. Define cells by gating dot plots of forward scatter (FSC) and side scatter (SSC).
   4. Measure the fluorescent intensity of anti-SSEA-1 monoclonal antibody and its isotype control on a "FL2-H" channel. Determine the fluorescent intensity of anti-Oct3/4 monoclonal antibody and its isotype control on a "FL3-H" channel. Quantify the fluorescent intensity of anti-SSEA-4 monoclonal antibody and its isotype control on a "FL4-H" channel.
   5. Measure the fluorescent intensity of gated events (over 90% of total events) in histograms with the percentages of subpopulations positive for a particular isotype control below 1%.

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