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Adipose tissue is an abundantly available source of proliferative and multipotent mesenchymal stem cells with exceptional potential for regenerative therapeutics. We previously demonstrated that both human and mouse adipose-derived stem (ADS) cells can be reprogrammed into induced pluripotent stem (iPS) cells with efficiencies higher than have been reported for other cell types. The ADS-derived iPS cells can be generated in a feeder-independent manner, representing a unique model to study reprogramming and a significant step toward establishing a safe, clinical grade of cells for transplantation. Here we provide a detailed protocol for isolation, preparation and transformation of ADS cells from fat tissue into iPS cells in a feeder- and xenobiotic-free process. This protocol also describes how ADS cells can be used as feeder cells for maintenance of other pluripotent stem cells. ADS derivation is rapid and can be completed in less than 1 week, with mouse and human iPS reprogramming averaging 1.5 and 2.5 weeks, respectively.
Stem cells are ideal and promising sources not only for studying cellular and developmental processes but also for developing regenerative therapeutics. However, many problems remain before these cells can serve as a practical source for clinical applications. The challenges include availability, efficiency, safety and ethical issues. Stem cells present in the human body are usually very limited in number and protocols to fully optimize efficiency of purification and use are still under development. These include development of particular cell lines capable of differentiating into clinically suitable cell types1, transplantation methods to deliver the cells effectively into desired locations, and introduction of personalized traits tailored for individual needs2. Safety is another concern because typical cell culture methods make use of animal sourced products such as serum and mouse feeder cell layers3. In addition, cells to be transplanted typically originate from heterologous sources, increasing the risk of immune rejection4. For example, commonly used human embryonic stem (ES) cells express an immunogenic non-human cell surface modification (sialic acid Neu5Gc), presumably due to the use of animal-derived products in the culture media5. Furthermore, ethical issues continue to be a matter of debate, especially for the use of human ES cells6. Many of these problems, we believe, can potentially be solved by using human adipose-derived stem (ADS; also known as processed lipo-aspirate, adipose stromal, or adipose tissue-derived mesenchymal stem) cells7.
In addition to mature adipocytes, adipose tissue contains relatively abundant progenitor and mesenchymal stem cell (MSC) populations in the stromal vascular fraction (SVF). It is estimated that as many as 1% of SVF cells are MSCs. In contrast, only 0.001 – 0.002% of cells in the bone marrow, which is considered a standard hub of adult stem cells, represent MSCs8. ADS and bone marrow-derived MSC cells are proliferative and ‘multipotent,’ having the capacity to differentiate into limited cell types such as adipocytes, osteocytes, chondrocytes, and myocytes9,10. MSCs from adipose and bone marrow possess many common cell surface markers though a precise definition of ADS cell markers is not well established11,12. It is advantageous to use fat tissue as a potential source for regenerative medicine because the tissue is abundant in this present era of global-wide obesity and relatively easy to obtain. Liposuction, a procedure to remove excess fat tissue, is among the most common plastic surgeries operated in the United States. Additionally, in contrast to bone marrow-derived MSCs that require initial plating with high density (>50,000 cells/cm2), ADS cells can be seeded and maintained as low as 3,000 cells/cm2.
The discovery of the ability to create ES cell-like, induced pluripotent stem (iPS) cells by transducing four transcription factors into somatic cells has revolutionized the stem cell field13–16. This technology enables not only the study of cell dedifferentiation and differentiation but also development of patient-specific cells for disease models and regenerative therapies. Unlike ES cells, which are isolated from the blastocysts of an embryo, iPS cells can be derived from adult somatic cells and thus avoid many ethical concerns. We recently investigated if the ‘multipotency’ of ADS cells can be upgraded to ‘pluripotency,’ by introducing the four standard reprogramming factors, Oct4, Sox2, Klf4 and c-Myc7. The resultant adipose-derived iPS cells exhibit all the characteristics and morphologies of ES cells from the corresponding species. Mouse and human adipose-derived iPS cells are over 5-fold and 100-fold more efficient in reprogramming than mouse and human fibroblasts, respectively, which are the most commonly used cell lines for iPS generation. Importantly, both mouse and human adipose cells are capable of giving rise to iPS cells without the need for feeder cells. This is due at least in part by the intrinsically high expression of self-renewal supporting factors such as basic FGF, vitronectin, fibronectin and LIF in ADS cells7. We found that ADS cells also have the ability to serve as feeders for other pluripotent stem cell lines. This is a key step toward establishing safe, clinical-grade human iPS cell lines because culture of iPS cells typically requires feeder layers of mouse embryonic fibroblasts (MEF) and use of media containing animal components such as serum and bovine serum albumin (BSA)3. We believe that generation of human adipose-derived iPS cells in xenobiotic-free conditions will obviate the development of non-human immunogenic surface markers described above. In addition, use of chemically defined media will ensure quality control, mitigate sources of biological variability and reduced the risk of animal-derived pathogen contamination.
Here we describe detailed protocols of ADS cell isolation and subsequent iPS cell line derivation. Use of ADS cells for supporting heterologous pluripotent stem cells as feeders is also described. In addition to retrovirus production of reprogramming factors, derivation of mouse ADS-derived iPS cells in feeder-free conditions is described, which produces about a 0.25% reprogramming efficiency. We also compare derivation of human ADS-derived iPS cells in feeder-dependent, feeder-free or xeno-free conditions. The reprogramming efficiencies are approximately 1.0% with feeder cells and ~0.008% without feeders or xenobiotics. The relative abundance and ease of isolation and derivation of ADS cells offers an ideal system for the study of the molecular mechanisms of cellular reprogramming and translation of stem cells into cell-based therapies.
C57BL/6J (Jackson Lab stock no. 000664) or Oct4-EGFP (Jackson Lab strain name B6;129S4-Pou5f1tm2Jae/J; stock no. 008214; isolated cells show green fluorescence upon successful reprogramming) Any work involving use of animals must be reviewed and approved by the Institutional Animal Care and Use Committee.
Immediately prior to the digestion, prepare a fresh solution of 2.5 µg/ml collagenase (wt/vol), 1% BSA, 50 µg/ml D-glucose and 200 nM adenosine in HBSS. Filter sterilize.
Prepare 154 mM NH4Cl and 20 mM Tris. Adjust pH to 7.4 and filter sterilize. Stable at room temperature for up to 4 weeks.
pMX vectors containing the cDNAs of mOct4 (Plasmid 13366), mSox2 (Plasmid 13367), mKlf4 (Plasmid 13370), mc-Myc (Plasmid 13375) (Addgene), hOct4 (Plasmid 17217), hSox2 (Plasmid 17218), hKlf4 (Plasmid 17219), hc-Myc (Plasmid 17220), retroviral gag–pol packaging plasmid (Plasmid 8449), VSV-G expression plasmid (Plasmid 8454) (Addgene), pMX-GFP (Cell Biolabs). Since retrovirus pseudotyped with VSV-G can infect humans, any procedure involving this virus must be performed under Biosafety Level 2 containment.
High-glucose DMEM, 10% FBS (vol/vol), 1× GlutaMAX, 1× NEAA, penicillin/streptomycin (100 U ml−1 and 100 µg ml−1, respectively). To prepare 500 ml of the media, mix 50 ml FBS, 5 ml GlutaMAX, 5 ml NEAA solution and 5 ml penicillin/streptomycin, and then fill up to 500 ml with DMEM. It can be stored at 4 °C for 4 weeks.
To increase growth of mADS cells, supplement complete DMEM media with 5 ng/ml basic FGF. Use of heat-inactivated FBS is recommended. Expansion media may be stored at 4 °C for up to 2 weeks.
While Expansion DMEM media is good for culturing human ADS cells, we found that Mesenchymal Stem Cell Growth Medium (PromoCell, cat. no. C-28010) achieves optimum growth for regular culture conditions. For xeno-free conditions, use StemPro MSC SFM XenoFree (Invitrogen, cat. no. A10675-01).
DMEM without phenol red, 2% heat inactivated FBS, and 0.1% sodium azide. It can be stored at 4°C for up to 4 weeks.
Add a 0.1% gelatin solution to coat the dish well, ensuring that the entire bottom surface is coated. Incubate for at least 30 min at 37 °C. Immediately before cell plating, aspirate the solution.
To prepare 500 ml of ESC media, mix 400 ml KO-DMEM, with 75 ml ES Cell Qualified FBS, 5 ml GlutaMAX, 5 ml Nucleotides, 5 ml penicillin/streptomycin, 5 ml NEAA solution, 5 ml diluted β-mercaptoethanol (100X) and 50 µl LIF (from 1 × 106 U/ml stock; 1,000 U/ml final). Store ESC media at 4 °C for up to 1 week.
To prepare 500 ml of ESC media, mix 400 ml KO-DMEM, with 75 ml KOSR, 5 ml GlutaMAX, 5 ml Nucleotides, 5 ml penicillin/streptomycin, 5 ml NEAA solution, 5 ml β-mercaptoethanol (100X) and 50 µl LIF (1,000 U/ml final). Store at 4 °C for up to 1 week.
To prepare 500 ml of hESC media, mix 385 ml of DMEM/F-12 base media with 100 ml knockout serum replacement, add 5 ml NEAA solution, 5 ml Glutamax, 5 ml β-mercaptoethanol (100X) and FGF2 to final concentration of 10 ng/ml (add 100 ul of a 50 ug/ml stock of FGF2). Sterlize by filtering through 0.22 µm vacuum filter unit. Make fresh and store at 4°C for up to 2 weeks.
Refer to the manufacturer’s recommendation on handling and thawing matrigel. It is important to thaw on ice and keep all components chilled that are to be used for aliquoting or diluting matrigel. We recommend aliquoting into 1 mg each (volume will depend on each production lot – see the product’s certificate of analysis for protein concentration). Store aliquots at −80°C. Dilute 1 mg of matrigel into 24 ml of ice-cold DMEM/F-12 media and mix by pipeting up and down. Immediately pipette 2 ml per well (6-well format) or 5 ml per 6-cm dish of the matrigel solution. Swirl to evenly coat surface. Allow product to gel and coat surface at 37°C for 1 – 48 h. Prepare for cell culture by first aspirating excess matrigel solution, then add sufficient volume of fresh hES cell media of choice to cover the surface. Return the plate/dish to the incubator to warm and CO2 equilibrate. Passage cells onto the prepared surface after 10 minutes and up to several hours.
Isolated ADS cells should proliferate well, with a typical doubling period of 2 to 3 days. ADS cells are positive for cell surface antigens Sca-1, CD29, CD73 and CD90, and negative for CD31 and CD45. The CD34 marker is present in the initial culture, but is quickly lost after one to two passages. ADS cells are phenotypically and functionally very similar to bone marrow-derived MSCs. The authenticity of the mesenchymal origin of ADS cells can be tested by differentiation of these cells into those of the mesodermal cell lineage (i.e. adipocytes, osteoblasts and chondrocytes) by the addition of well-defined standard adipogenesis, osteogenesis and chondrogenesis cocktails, respectively19,20,17.
Mouse iPS cells can be produced from ADS cells with an efficiency range of 0.1 – 0.5%. The presence of MEF feeders during iPS derivation does not affect the percentage of emerging colonies, but appears to better support the maintenance of self-renewal of many colonies long term. Mouse ADS-derived iPS cells can be stained for pluripotent markers including alkaline phosphatase, Nanog and SSEA1. At least several good colonies of iPS cells need to be maintained and stored. Mouse iPS cells can be further characterized for their pluripotency by in vitro differentiation through embryoid body (EB) formation (1 – 2 weeks), in vivo differentiation through teratoma formation (2 – 4 weeks), and/or generation of chimeric mice through injection into blastocyst (1 – 2 months).
The efficiencies of human iPS cell derivation are around 0.5 – 1.5% in the feeder-dependent condition, and around 0.008% in the feeder-free and xeno-free conditions. However, efficiencies are highly affected by many variables such as retrovirus titer, transduction efficiency, cell viability and proliferation states, choice of media, growth factors, matrix and feeder cells, etc. It is best to pick and maintain as many colonies as possible to ensure the derivation of iPS cells that have the same morphology and gene/protein expression profiles as the ES cells. It is important to karyotype the cells to check for any chromosomal abnormalities after extended passages. Common pluripotency tests of human iPS cells are positive staining for alkaline phosphatase activity or pluripotency markers including SSEA3, SSEA4, TRA-1-60 and TRA-1-81 (1 day), in vitro differentiation through EB formation (2 – 4 weeks) and in vivo differentiation assays through teratoma formation (4 – 8 weeks) by injecting cells subcutaneously into immunodeficient animals (e.g. SCID mice).
We thank J.M. Gimble for helpful discussion, L. Ong and S. Ganley for administrative assistance, and R. Yu for advice and editing of the manuscript. This work was supported by grants from the National Institutes of Health (HD027183, DK057978, and DK062434), California Institute for Regenerative Medicine (RB2-01530), and Howard Hughes Medical Institute.
AUTHOR CONTRIBUTIONSS.S. and Y.K. designed and performed the experimental procedures. S.S., Y.K., and W.T.B. wrote the protocols. R.M.E. is the project leader, obtained funding, reviewed and edited the protocols.