Infrapatellar fat pad mesenchymal stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.
The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.
Mesenchymal stem cells (MSCs) are an attractive candidate for cell-based therapies in regenerative medicine1,2. They can be harvested from a variety of tissue sources such as bone marrow, umbilical cord, placenta, dental pulp, and subcutaneous adipose tissue3. However, as the availability of stem cells in adults is limited and their isolation procedure is often invasive (resulting in donor site morbidity), it is desirable to have an alternative stem cell source that could circumvent these challenges.
The knee joint is a depot of various cell types, such as infrapatellar fat pad-derived MSCs, synovial membrane-derived MSCs, synovial fluid-derived MSCs, ligament fibroblasts, articular chondrocytes, etc4,5,6. These cells have the potential to be widely explored in musculoskeletal tissue engineering-based research. Therefore, the knee joint could be a possible and reliable source of multiple types of MSCs. Adipose depot located in the knee joint, known as the infrapatellar fat pad (IFP) or Hoffa's fat pad, is a promising and alternative choice of MSC depot. The IFP is a relatively easily accessible and clinically obtainable source of MSCs, as it is routinely resected and discarded as surgical waste during knee arthroscopy or open knee surgery. Removal of the IFP is associated with minimal donor-site morbidity, which also makes it an attractive tissue source. While having a similar phenotypic profile, MSCs from IFP (IFP-MSCs) have enhanced clonogenic potential when compared with bone marrow-derived mesenchymal stem cells (BM-MSCs)6 and better proliferative capacity compared to subcutaneous adipose-derived stem cells (ADSCs)7. Interestingly, compared to synovial fluid-derived MSCs (SF-MSCs), IFP-MSCs do not lose their proliferative capacity at late passages, nor does doubling time increase at late passages. This suggests that, during cell expansion, IFP-MSCs can achieve a sufficiently large number of cells for in vitro tissue engineering applications without compromising their proliferation rate8. Recent studies have also suggested that IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived MSCs (ADSCs), probably due to their anatomical proximity to articular cartilage, indicating their suitability for cartilage tissue engineering6,7,9,10. Moreover, they also possess age-independent osteogenic differentiation potential11. Intra-articular injection of IFP-MSCs has been shown to reduce pain and improve knee joint functions in patients with osteoarthritis (OA)12,13. Further, strong immunosuppressive responses and improved immunomodulatory properties of IFP-MSCs in the presence of inflammatory cytokines during pathological conditions have also been reported6.
IFP-MSCs are a promising and alternate source of MSCs; however, their therapeutic benefit in tissue engineering and regenerative medicine is relatively less explored. The existing studies on IFP-MSCs have majorly utilized cells from human donors. Amongst these, a few recent studies have investigated IFP-MSCs from healthy human donors (non-arthritic patients, aged 17-60 years)6,14, whereas most of the studies have used IFP-MSCs from aged patients undergoing total knee replacement surgery (diseased patients, age 70-80 years). As both age and disease are known to alter the normal functioning of stem cells (reduced number and loss of functional potential), this could potentially lead to inconsistencies in the outcome of the MSC-based studies7,15,16,17. In addition to that, the use of IFP-MSCs from patients with pathophysiological conditions (e.g., arthritis and obesity) also poses difficulty for understanding the basic characteristics of healthy cells in vitro, thereby acting as a limiting factor in the development of MSCs-based therapies. To overcome these issues, the use of IFP-MSCs from healthy donors is vital. As access to a healthy human donor is challenging, animal models could be a better alternative. In this regard, there are a few studies where IFP has been isolated from mice18. However, owing to the small size of the fat pad in normal mice, fat tissues from multiple animals have been combined to get enough tissue to execute elaborate experimental procedures19. Hence, there is a need for a large animal model, which could fulfill the requirement for the higher number of cells and simultaneously comply with the 3R principles (refine, replace, and reduce) in animal research20. The usage of large animals has significant implications in translational research. Specifically, in musculoskeletal tissue engineering, a range of large animals such as dogs, pigs, sheep, goats, and horses have been investigated21. Goat (Capra aegagrus hircus) is an excellent choice of large animal since its stifle joint has the closest anatomy to the human knee joint22,23,24. The subchondral bone trabecular structure and subchondral bone thickness of goats are similar to humans, and the proportion of the cartilage to bone is also reported to be close to humans21. In addition, goats have been widely domesticated throughout the world, making them easily available when they are skeletally mature. Further, low maintenance costs and easy handling have made them an attractive animal model for research22.
In the present study, a simple protocol for the isolation of IFP-MSCs from the stifle joint of Capra aegagrus hircus (goat) and in vitro culture conditions for their expansion and differentiation are demonstrated. The isolated cells are adherent, have MSC-like morphology, form CFU-F (colony-forming unit-fibroblast) colonies, and possess adipogenic, chondrogenic, and osteogenic differentiation potential. Therefore, IFP-MSCs show potential as an alternative source of MSCs for biomedical applications.
The protocol is based on the isolation of IFP-MSCs from goats. Goat IFP and blood were collected from a local abattoir. Since such tissue collections are outside the purview of an Institutional Animal Ethics Committee, ethical approval was not required.
1. Isolation of IFP-MSCs from the femorotibial joint of goat knee
2. Maintenance and expansion of isolated cells
3. Evaluation of the clonogenic ability of IFP-MSCs using colony forming assay (CFU-F)
4. Differentiation potential of IFP-MSCs
Isolation of IFP-MSCs from the femorotibial joint of goat
The steps involved in the isolation of IFP-MSCs from the stifle joint of a goat are depicted in Figure 1. The fat pad present in the inner non-articulating surface of the patella was removed, minced, and enzymatically digested. The IFP-MSCs were successfully isolated and cultured in vitro (Figure 2A).
Expansion and clonogenic ability of IFP-MSCs
The isolated cells were cultured in vitro in expansion media. The cells started adhering to the tissue culture plate within 12 h of seeding and were mostly round in shape at day 0. They were homogenously adherent to the plate and attained elongated morphology within 24 h. This morphology was maintained throughout the duration of the culture. The cells proliferated efficiently in culture and became 80%-90% confluent within 6 days of expansion (Figure 2A). Additionally, the isolated cells displayed clonogenic capacity, assessed by colony-forming unit-fibroblast (CFU-F) assay (Figure 2B). These results indicate that the isolated cells have efficient proliferative and self-renewal capabilities in vitro.
Differentiation potential of IFP-MSCs
To characterize whether the isolated cells were multipotent and possessed characteristic features of MSCs, they were induced to differentiate into multiple lineages. The isolated cells, when induced towards adipogenic lineage, produced lipid droplets, as can be observed from the brightfield images (Figure 3A). This was further confirmed by Oil Red O staining on day 14 and day 21, suggesting the ability of these cells to differentiate into adipocytes. On the other hand, no visible oil droplets were observed in the cells cultured in the absence of adipogenic-inducing factors (Figure 3B,C).
To determine the chondrogenic potential of the isolated cells, the cells were encapsulated in plasma hydrogel and were allowed to differentiate into the chondrogenic lineage. As depicted from the gross images of the plasma hydrogel (Figure 4A), the neotissue formed after 14 days in the induced group appeared white and glossy (similar to articular cartilage), whereas in the uninduced group, it was pale white, and reduced glossiness was observed. Additionally, the cells in the induced group were able to secrete one of the major components of the cartilage matrix (i.e., sulfated glycosaminoglycans), evidenced by positive histological staining for Alcian Blue (Figure 4B) and Safranin O (Figure 4C). In contrast, the hydrogel sections from the uninduced groups were negative for Safranin O and Alcian Blue staining. These results collectively indicate the chondrogenic differentiation potential of the MSCs in the presence of chondrogenic stimuli only.
The isolated cells also demonstrated osteogenic differentiation potential. After 14 days and 21 days in culture, no spontaneous mineralization was observed (uninduced group). However, when induced with osteogenic media, mineralization was evident by the positive staining for alkaline phosphatase (ALP) at the end of 14 days (Figure 5A). Moreover, after 28 days of culture in osteogenic media, calcified depositions were visualized by positive Alizarin Red-S staining (Figure 5B). These results demonstrate that the isolated cells from goat infrapatellar fat pad, when induced, have the potential to differentiate into adipogenic, chondrogenic, and osteogenic lineages.
Figure 1: Schematic for the isolation of IFP-MSC from the stifle joint of goat knee. Step 1. Collect the goat knee sample from the slaughterhouse and proceed with the dissection in a biosafety cabinet; Step 2. Carefully examine the anatomy of the tissue and remove the surrounding muscles and adipose tissue; Step 3. Without disturbing the joint capsule, remove the muscles and adipose tissues to completely expose both the long bones (femur and tibia); Step 4. Make an incision in the synovium membrane and cut open the articulating joint to expose the patella and trochlear cartilage; Step 5. Remove the patella from the articulating joint carefully, without disturbing the infrapatellar fat pad (IFP), and keep it on a petri dish containing PBS; Step 6. Slowly remove the entire fat pad from the patella and keep it in a fresh petri dish for mincing and enzymatic digestion. (a. Femur, b. Tibia, c. Patella, d. Trochlear cartilage, e. Infrapatellar fat pad). Please click here to view a larger version of this figure.
Figure 2: Morphology and clonogenic potential of IFP-MSCs. (A) Micrographs were taken at passage 3 using an inverted brightfield microscope at 10x magnification. Immediately after seeding (day 0), the cells remained round in shape, at day 3 most of the cells attained elongated morphology, and on day 6 the IFP-MSCs became 80%-90% confluent (Scale bar = 100 µm). (B) Representative pictures (n = 3) of CFU-F colonies stained with crystal violet dye after 12 days of seeding. Please click here to view a larger version of this figure.
Figure 3: Adipogenic differentiation of IFP-MSCs. The upper panel indicates micrographs of uninduced IFP-MSCs, and the lower panel represents IFP-MSCs induced into the adipogenic lineage. (A) Representative brightfield images showing the formation of lipid vacuoles during adipogenesis (Scale bar = 100 µm). Representative images of Oil Red O staining of lipid droplets at (B) day 14 and (C) day 21 of differentiation (Scale bar = 20 µm). Please click here to view a larger version of this figure.
Figure 4: Chondrogenic differentiation of IFP-MSCs. The upper panel indicates hydrogel constructs/sections from uninduced hydrogel, and the bottom panel represents hydrogel subjected to chondrogenesis. (A) Gross images of plasma hydrogel constructs after 14 days in culture in the absence (uninduced) or presence (induced) of chondrogenic media supplemented with TGF-β1. Representative images of 10 µm sections of hydrogel stained with sGAG binding dye (B) Alcian Blue (blue) and (C) Safranin O (red) (Scale bar = 100 µm). Please click here to view a larger version of this figure.
Figure 5: Osteogenic differentiation of IFP-MSCs. The upper panel indicates uninduced cells, and the bottom panel indicates cells induced with osteogenic media. Micrographs and representative images of IFP-MSCs stained with (A) BCIP-NBT (alkaline phosphatase) after 14 days and (B) Alizarin Red-S (calcium deposition) after 28 days of differentiation (Scale bar = 100 µm). Please click here to view a larger version of this figure.
In the present protocol, a simple, reliable, and reproducible method for the isolation of MSCs from goat IFP has been provided. Cells isolated using this method have been successfully used in our previous studies for in vitro tissue regeneration. It was observed that the isolated cells were proliferative, were responsive to various growth factors, and retained their biological activity when seeded on electrospun fibers and scaffolds25,26. Moreover, it was observed that a large number of high-quality MSCs can be obtained and co-cultured with chondrocytes in injectable hydrogels to engineer articular cartilage in vitro27,28,29. While the procedure for the dissection and isolation of cells is simple, there are a few steps that need to be carefully followed for the successful isolation and expansion of MSCs from IFP. Firstly, since it is not possible to maintain truly aseptic conditions while bringing the sample from the slaughterhouse, it is advised to wipe the outer tissue with 70% ethanol to sterilize the tissue before starting the isolation procedure. The next important step is dissecting the fat pad from the knee joint. The IFP is located below the patella in the knee joint. The proximal end of the IFP is attached to the distal end of the patella30,31. Hence, it becomes very important to identify the correct location of the patella while performing the isolation procedure. Next, as IFP articulates with the trochlear cartilage in the femur and is covered with the synovium membrane30,31, making an incision to the synovium membrane is necessary to cut open the knee joint. It is also crucial to observe the overall health of the collected infrapatellar fat pad. As the IFP is composed of adipocytes (cells) and adipose connective tissue, such as collagen and glycosaminoglycans, the tissue is minced and digested using collagenase enzyme to release the cells9,32. The floating adipocytes are separated by centrifugation at a slow speed to have a homogenous culture of MSCs. The remaining adipocytes in the stromal vascular fraction must be removed from the tissue culture plate at each media change by washing the cell monolayer with PBS before adding fresh media. This step needs to be continued until all the visible adipocytes are removed. It is suggested to reseed the cells in a new plate after P0 cells reach confluency to get rid of the remaining adipocytes, which might have adhered to the plate wall and are difficult to remove. Finally, as the goat samples are mostly obtained from a slaughterhouse, it is difficult to trace the exact age of the goat, and, therefore, while isolating the cells, variation between samples needs to be carefully observed and documented. It is suggested to characterize the cells for self-renewal (CFU-F), proliferation, and differentiation potential after each isolation before they are used for various applications and mechanistic studies. Due to the requirement of a large number of cells in cell-based tissue engineering applications, the culture conditions that facilitate proliferation, delay senescence, and maintain the stemness of cells become centrally important. It was observed that MSCs cultured in 2-Phospho-L-ascorbic acid trisodium salt (PAA) and basic fibroblast growth factor (bFGF) showed a ~42-fold increase in their numbers compared to MSCs cultured in bFGF alone. In addition, the co-treatment of PAA and bFGF reduced the production of reactive oxygen species (due to their antioxidant properties) and senescence in stem cells. Therefore, the use of PAA during in vitro expansion of MSCs is desirable33.
While enzymatic digestion of tissues is universally employed and is an efficient method for isolating MSCs, the use of enzymes such as collagenases is time-consuming and expensive34. In the present study, the complete digestion of the tissue using collagenase took 12-16 h (overnight), which might lead to a decreased viability or altered surface phenotype of cells35. Hence, the method can further be optimized to reduce the duration of collagenase treatment for tissue digestion. Alternatively, an enzyme-free explant culture method that is used for the isolation of adipose-derived MSCs from lipoaspirates or inguinal fat pads can also be explored for the isolation of IFP-MSCs34,36,37.
IFP-MSCs, due to their ability to differentiate into chondrogenic, adipogenic, and osteogenic lineages possess wide therapeutic potential. Previous reports have demonstrated that IFP-MSCs exhibit better chondrogenic potential than other stem cells6,7,9,10. Hence, IFP-MSCs have gained interest and are considered a better candidate for cell-based therapy for the repair of cartilage defects and alleviating cartilage degradation in osteoarthritis. In a preclinical study, co-delivery of IFP-MSCs with cartilage extracellular matrix components through intraarticular injection resulted in enhanced cartilage regeneration in osteochondral defects38. Similarly, intra-articular injection of IFP-MSCs in the rabbit osteoarthritis (OA) model led to a decrease in the following disease parameters: cartilage degradation, osteophyte formation, subchondral bone thickening, and synovitis39. Moreover, results from a previously reported randomized clinical trial13 and a recently published open level phase 1 clinical trial40 have demonstrated that intra-articular injections of IFP-MSCs in patients with knee osteoarthritis are safe and result in the reduction of pain and improvement of joint function, indicating their promising therapeutic potential in ameliorating knee OA-related complications. IFP-MSCs also have been shown to develop augmented immunomodulatory properties in the presence of proinflammatory cues by facilitating the degradation of an inflammatory regulator, Substance P (SP), leading to a reduction in proinflammatory and catabolic molecules and, as a consequence, improved treatment of inflammation and fibrosis of synovium and IFP6. More importantly, IFP-MSCs processed in regulatory-compliant conditions showed better proliferation, differentiation capacity, and immunomodulatory properties compared to standard in vitro culture conditions, which demonstrates their potential for clinical success when used for mesenchymal stem cell (MSC)-based therapy41.
In conclusion, the present protocol describes the isolation of infrapatellar fat pad (IFP)-derived mesenchymal stem cells (MSCs) from goat stifle joint by enzymatic digestion and their maintenance in culture. The isolated cells have proliferative and self-renewal potential and can differentiate into adipogenic, osteogenic, and chondrogenic lineages. IFP-MSCs are a promising source of MSCs and have translational potential for regenerative medicine applications.
The authors have nothing to disclose.
SH acknowledges support from the Institute Post-Doctoral Fellowship of IIT Kanpur and SYST grant from DST (SEED Division) (SP/YO/618/2018). AM acknowledges the Indian Institute of Technology-Kanpur (IIT-Kanpur) for an Institute fellowship. DSK acknowledges Gireesh Jankinath Chair Professorship and Department of Biotechnology, India, for funding (BT/PR22445/MED/32/571/2016). AM, SH, and DSK thank The Mehta Family Centre for Engineering in Medicine at IIT-Kanpur for their generous support.
β-glycerophosphate | Sigma-Aldrich | G9422-10G | 10 mM |
0.25% Trypsin- 0.02% EDTA | Hi-Media | TCL049 | |
15-mL centrifuge tube | Corning | ||
2-Phospho-L-ascorbic acid trisodium salt | Sigma | 49752-10G | 50 µg/mL |
2-Propanol | Sigma-Aldrich | I9516 | |
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) | HiMedia | TCL021-50ml | 10 mM |
50-mL centrifuge tube | Corning | ||
Alcian Blue | Hi-Media | RM471 | For sufated gycosaminoglycans staining |
Alizarin Red S | S D Fine-Chem Limited | 26048-25G | For calcium deposition |
Amphotericin B | HiMedia | A011 | 2.5 µg/mL |
Basic fibroblast growth factor (bFGF) | Sino Biologicals | 10014-HNAE | 5 ng/mL |
BCIP/NBT ALP Substrate | Sigma | B5655-5TAB | For ALP staining |
Biological safety cabinet | |||
BSA | HiMedia | MB-083 | Long name: Bovine Serum Albumin (1.25 mg/mL ) |
Cell strainer | HiMedia | TCP-182 | 70 µm |
Centrifuge | REMI | ||
Ciprofloxacin | RANBAXY LAB. Limited | B17407T1 | 2.5 µg/mL |
Crystal Violet | S D Fine-Chem Limited | 42555 | |
D(+)-glucose | Merck | 1.94925.0521 | 25 mM |
Dexamethasone | Sigma-Aldrich | D2915 | 1 µM |
DMEM LG | SIGMA | D5523 | Long name: Dulbecco’s Modified Eagle’s Media Low Glucose |
Ethanol | Merck | 100983 | |
FBS | Gibco | 10270 | Long name: Fetal Bovine Serum |
Formaldehyde solution 37%-41% | Merck | 61780805001730 | |
Indomethacin | Sigma-Aldrich | I7378 | 100 µM |
Insulin | Sigma-Aldrich | I9278 | 10 µg/mL |
Inverted microscope | Nikon Eclipse TS 100 | ||
ITS + 1 | Sigma-Aldrich | I2521-5mL | Long name: insulin, transferrin, sodium selenite + linoleic-BSA |
L-Proline | HiMedia | TO-109-25G | 1 mM |
Magnesium chloride | Merck | 61751605001730 | For lysis buffer |
Methanol | Meck | 1.07018.2521 | |
Micropipettes and sterile tips (20 µL, 200 µL, 1000 µL) | Thermoscientific | ||
MUSE Cell analyser | Merck Millipore | For cell counting | |
OCT compound | Tissue-Tek | 4583 | Long name: Optimal Cutting Temperature |
Oil Red O dye | S D Fine-Chem Limited | 54304 | For lipid vacuole staining |
Penicillin-Streptomycin | HiMedia | A007 | 100 U/mL |
Petri dishes (150 mm and 90 mm) | NEST | ||
Safranin O | S D Fine-Chem Limited | 50240 | For sufated gycosaminoglycans staining |
Sodium citrate | Sigma-Aldrich | C3434 | 3.4 % (w/v) |
Sterile scissors, forceps and scalpels | For isolation of IFP-MSC | ||
Sucrose | Merck | 1.94953.0521 | 35 % (w/v) |
TGF-β1 | Sino Biologicals | Long name: Transforming growth factor- β1 (10 ng/mL) | |
Tissue culture incubator 37 °C, 5% CO2 | Thermoscientific | ||
Tris buffer | Merck | 61771405001730 | For lysis buffer |
Triton X100 | S D Fine-Chem Limited | 40632 | For lysis buffer |
Type II collagenase | Gibco | 17101015 | 1.5 mg/mL |
Vitamin D3 | Sigma | C9756-1G | 10 nM |
Well plates (6 -WP and 24-WP) | NEST |