This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.
Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.
Obesity, present in about 30% of the population in the USA, with a body mass index over 30, has emerged as a prevalent worldwide phenomenon1. Obesity tends to lead to related complications including cardiovascular diseases, type-2 diabetes, and cancer2-4. Therefore, dealing with obesity is an important priority. Obesity is manifested by massive expansion of adipose tissues, and is attributed to excessive food consumption and a sedentary life style in modern society. Hence, deciphering the transcriptional regulation of adipogenesis and lipogenesis could hold promise to treat obesity or diabetes5.
The 3T3-L1, 3T3-F442A and other mouse adipogenic cell lines have been applied to study adipogenesis or lipogenesis during adipose tissue development. However, there are some discrepancies in regulatory mechanisms between cell lines in vitro and animals in vivo6. Primary adipose-derived stem cells (ADSC) in the stromal-vascular cell fraction can be isolated directly from white adipose tissues and induced to differentiate. Differentiation of ADSC into adipocytes most likely recapitulates the process of adipogenesis and lipogenesis in adipose tissue development in vivo7.
Pigs are a suitable animal model for studying adipogenesis and lipogenesis in adipose tissue development. Our previous porcine studies8-10 demonstrate that expression of sterol regulatory element-binding transcription factor 1c (SREBP1c), an important transcription factor known to modulate transcription of lipogenic fatty acid synthase, is inhibited by polyunsaturated fatty acids (PUFA) in the porcine liver and adipose tissues. The expression of porcine SREBP1c decreased by PUFA in vivo and in vitro is similar to other species such as humans and mice11-13. These pig studies in vitro are primarily in differentiated adipocytes derived from porcine ADSC (pADSC). Therefore, this primary cell culture of pADSC can be used to serve as a reliable cellular system to study adipose tissue development or other stem cell applications.
Note: This method has been established and used in research reported previously14-17 from this laboratory; over time the methodology was modified. The current procedure was performed using an average of 60 g of porcine subcutaneous adipose tissues from one piglet (7 to 9 days old) with seeding on 6-well tissue culture plates. All procedures were performed at RT unless otherwise designated. All the animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at National Taiwan University.
1. Prepare Digestion Medium
2. Dissect Subcutaneous Adipose Tissues from Pigs
Figure 1. A customized slicer used for isolation of pADSC. Dissected adipose tissues from porcine dorsal subcutaneous adipose tissues are composed of the fat layer with attached skin layers. A slicer is required to slice the fat layer approximately 1 mm thick with avoidance of over-slicing into the skin layers. From left to right: slice holder, slicer pad, carbon steel slicer blade, and screws. Slicer blade is inserted between slice holder and slicer pad when assembled. Please click here to view a larger version of this figure.
3. Collection of pADSC from the Stromal-vascular Fraction
4. Identification Stem-cell Surface Markers of pADSC by Flow Cytometry
5. Differentiation of pADSC into Adipocytes, Osteocytes and Chondrocytes
6. Staining of Differentiated Adipocytes, Osteocytes and Chondrocytes
The pADSC derived from pig dorsal subcutaneous fat were seeded on the culture plates or dishes and shown in Figure 2. The morphology of the pADSC derived from the stromal-vascular fraction is similar to mouse or human ADSC. Twenty-four h after seeding, subconfluent pADSC are adhered and have an expanded fibroblast-like morphology (Figure 2A). The pADSC will become confluent within 72 h and are ready for adipocyte or other mesenchymal-type differentiation (Figure 2B). pADSC exhibit strong adipogenic potential after chemical induction and mature adipocytes can be observed after 9 days of differentiation with over 90 % of the pADSCs showing adipogenic differentiation (Figure 2C).
To address the characteristics of pADSC derived in this protocol, surface markers of pADSC were evaluated by flow cytometry analysis. As shown in Figure 3, surface markers for mesenchymal stem cells, including CD29, CD44, CD90 and MHC I (or HLA I), were highly expressed. Negative surface markers, such as CD4a, CD31, CD45 and MHC II (or HLA II) were barely detectable in pADSC derived in the protocol (Figure 3). These results demonstrate that these pADSC exhibit mesenchymal-type stem cell characteristics without significant endothelial or haematopoietic stem cell contamination, including myeloid or lymphoid progenitors.
To further confirm that pADSC represent mesenchymal stem cells, the multipotency of pADSC was examined by differentiation into adipocytes, osteocytes and chondrocytes. These adipocytes, osteocytes and chondrocytes were stained by specific dyes, Oil Red O, Alizarin Red S, and Toluidine Blue O, respectively (Figure 4). These data indicate that this protocol generated pADSC that retained multipotency with full characteristics resembling mesenchymal-type progenitors.
Figure 2. Morphology of pADSC from porcine back fat region. (A) Subconfluent pADSC adhered and expanded after 24-h seeding on a 6-well culture plate. (B) pADSC became confluent after 72-h seeding on a 6-well culture plate. (C) Mature adipocytes were observed after 9 days of adipogenic differentiation from pADSC. Images were taken at 100 x magnification using phase contrast microscopy. Please click here to view a larger version of this figure.
Figure 3. Identification of stem-cell surface markers for pADSC. 1 x 105 of pADSC were reacted with specific antibodies and analyzed for stem cell markers by flow cytometry analysis. Numbers indicate the percentage of stained cells in the population (red) compared with the unstained control. The x-axis represents the relative fluorescence intensity. The y-axis represents the population of cells. Please click here to view a larger version of this figure.
Figure 4. Multipotent differentiation of pADSC. Multipotency of pADSC was determined by differentiating pADSC into (A) adipocytes, (B) osteocytes (C) chondrocytes and stained by representative dyes, Oil Red O, Alizarin Red S, and Toluidine Blue O, respectively. Images were taken at (A) 100 x, (B) 200 x, and (C) 100 x magnification using phase contrast microscopy, respectively. Please click here to view a larger version of this figure.
Here we present a reliable cellular system to study adipose tissue development in primary cell culture of pADSC. Compared to other immortalized cell lines, this method provides a convenient way to isolate large quantities of high quality adult mesenchymal stem cells that can be applied to study differentiation processes of adipocytes or other mesenchymal lineages related to animal development in vivo. The critical modified step in this protocol is that we derive pADSC using a 7- to 9-day old piglet because it is easy to handle the small piglet compared to older pigs and similar to other species19,20, the yield and multipotency of pADSC decreases as the pig ages21.
Potential stem cell sources include embryonic stem cells (ESC), induced pluripotent stem cells (iPSC), and postnatal adult stem cells. The constraint of ADSC, classified as adult multipotent stem cells, is that multipotency of adult stem cells in differentiating divergent lineages is relatively limited compared with ESC or iPSC. However, ethical issues regarding derivation of ESC and oncogenic properties of iPSC restrain the application of ESC and iPSC22,23. Therefore, numerous investigators have focused on adult stem cells with efforts to enhance pluripotency. The most common source of adult mesenchymal stem cells (MSC), which has long been studied, is bone marrow-derived mesenchymal stem cells24. However, harvesting bone marrow is considered a relatively painful procedure. Another concern is that the yield of stem cells from the bone marrow is finite. Bone marrow aspirates yield an average of 6 × 106 nucleated cells per ml, and MSC only represent 0.001 to 0.01% of all the nucleated cells. After considering these drawbacks, ADSC is suggested as a less obtrusive source to obtain multipotent stem cells25,26.
Limitations on the use of ADSC in regenerative medicine are dependent, to a large extent on cell yield and quality. Therefore, the significance of employing pigs to isolate ADSC in this protocol is to yield a large quantity of high quality adult stem cells. The pig is a useful animal model representing humans because of the comparable organ size and many physiological and biochemical similarities between the species27-30. Acquiring hADSC from commercial companies is expensive and in many cases the cells have been manipulated, passaged or cryopreserved. Acquiring human clinical samples is relatively difficult because of ethical issues and production of ADSC is limited. We derive approximately 6 x 105 hADSC per g fat after collagenase digestion. With 100 g of female breast adipose tissue (an average sampling), a total of 6 x 107 cells can be harvested. Using an individual mouse, the yield is even more limited. A total of 1 x 106 cells can be harvested from 0.4 g of subcutaneous mouse inguinal adipose tissue from both legs of an adult FVB mouse (6-8 weeks old). However in one individual pig (7 to 9 days old), a total of 2 x 108 cells can be easily harvested from 60 g of subcutaneous adipose tissue obtained from the dorsal fat depot. The pADSC derived in this protocol have full mesenchymal-type multipotency and appropriate mesenchymal stem cell markers. Therefore, pADSC are a favorable source to obtain large quantities of adult stem cells without compromising stem cell quality.
The application of pADSC is not restricted to deciphering adipocyte differentiation including adipogenesis and lipogenesis. Recently, ADSC have become a popular source of stem cells in the field of regenerative medicine22,31,32. Compared to other stem cell sources, ADSC retain a unique advantage of being easily accessible and abundant, and their robust multipotency has been demonstrated to be a promising source for stem cell therapy and tissue engineering22,33,34. The easy accessibility of adipose tissue makes ADSC one of the least intrusive ways to get multipotent progenitors. Recently, we differentiated pADSC into glucose-responsive insulin-secreting clusters, indicating that pADSC are not limited to mesenchymal differentiation (unpublished data). Others have also been demonstrated that ADSC can be differentiated into many cell types derived from other germ layers such as endodermal hepatocytes (from hADSC35 or pADSC36) or ectodermal neurons (from hADSC37 or pADSC38). Thus, pADSC could be used for high-throughput drug or biomaterial screening by directing cells to divergent differentiation processes to yield desired lineages. Therefore, pADSC derived in this protocol have potential application in stem cell therapy and tissue transplantation for regenerative medicine research.
The authors have nothing to disclose.
The authors would like to express gratitude to all the lab members for the extensive discussion and technique supports in this protocol. Research performed in the lab was supported by grants from Ministry of Science and Technology (MOST 103-2314-B-002-126 and MOST 102-2313-B-002-026-MY3) and by grants from Aim for the Top University Plan (104R350144) of the National University, Taiwan.
Reagents | |||
Collagenase, Type II | Sigma-Aldrich | C6885 | |
DMEM, high glucose, pyruvate | Life Technologies | 11995-040 | |
DMEM/F-12, HEPES | Life Technologies | 11330-032 | |
Fetal Bovine Serum (FBS) | Biological Industries | 04-001-1 | |
Penicillin-Streptomycin-Amphotericin B (P/S/A) solution | Biological Industries | 03-033-1 | For antibiotics and antimycotic usage |
αMEM, no nucleosides | Life Technologies | 12561-049 | |
ACK lysis buffer | Life Technologies | A10492-01 | |
Trypsin-EDTA (0.25%), phenol red | Life Technologies | 25200072 | |
CD4a-PE | Sigma-Aldrich | SAB4700063 | |
CD29-PE | Sigma-Aldrich | SAB4700398 | |
CD31-PE | Sigma-Aldrich | SAB4700467 | |
CD44-PE | Sigma-Aldrich | SAB4700183 | |
CD45-PE | Sigma-Aldrich | SAB4700483 | |
CD90-PE | Sigma-Aldrich | SAB4700686 | |
HLA Class I-PE (MHC I) | Sigma-Aldrich | SAB4700640 | |
HLA-DR-PE (MHC II) | Sigma-Aldrich | SAB4700662 | |
Insulin | Sigma-Aldrich | I9278 | |
3,3',5-Triiodo-L-thyronine (T3) | Sigma-Aldrich | T6397 | |
Transferrin | Sigma-Aldrich | T2036 | |
3-isobutyl-1-methylxanthine (IBMX) | Sigma-Aldrich | I7018 | |
Dexamethasone | Sigma-Aldrich | D4902 | |
Rosiglitazone | Cayman | 71740 | |
β-Glycerophosphate | Sigma-Aldrich | G9422 | |
2-Phospho-L-ascorbic acid | Sigma-Aldrich | 49752 | |
TGFB1 Recombinant Human Protein | R&D Systems | 240-B-002 | |
Oil Red O | Sigma-Aldrich | O0625 | |
Alizarin Red S | Sigma-Aldrich | A5533 | |
Toluidine Blue O | Sigma-Aldrich | 198161 | |
Name | Company | Catalog Number | Comments |
Equipment | |||
Carbon Steel Blades | Thomas Scientific | 6727C18 | |
Falcon 100 µm cell strainer | Corning | 352360 | |
Falcon 6-well plate | Corning | 353046 | |
Falcon 100 mm dish | Corning | 353003 |