Despite the natural human aging process, the prevalence and functional activity of stem cells are highly preserved.
For years we have been stating “Age makes no difference” – We have published a small study confirming this.
In April 2017, a larger study has been released that confirms the stem cells collected from your body fat are still just as good whether you are young or old. The publication details are just below.
The progressive decline in tissue mechanical strength that occurs with aging is hypothesized to be due to a loss of resident stem cell number and function. As such, there is concern regarding use of autologous adult stem cell therapy in older patients. To abrogate this, many patients elect to cryopreserve the adipose stromal-vascular fraction (SVF) of lipoaspirate, which contains resident adipose stem cells (ASC). However, it is not clear yet if there is any clinical benefit from banking cells at a younger age.
We performed a comparative analysis of SVF composition and ASC function from cells obtained under GMP conditions from the same three patients with time gap of 7 to 12 years.
SVF, cryobanked under good manufacturing practice (GMP) conditions, was thawed and cell yield, viability, and cellular composition were assessed. In parallel, ASC proliferation and efficiency of tri-lineage differentiation were evaluated.
To assess adipogenic potential of cultured cells were lifted, counted and plated at 60,000 cells/cm2 in triplicate in both 12-well and 48-well plates. Cells were allowed to adhere overnight and then culture media was removed and replaced with StemPro adipogenesis differentiation media (Invitrogen #A10070-01) for 14 days, changing every 2 to 3 days. Control cells were identically cultured with basal media that did not contain adipogenic supplements, also in triplicate. To quantify lipid inclusions, fixed cells in 48-well plates were treated with 10 µg/mL Nile Red (Molecular Probes #N1142) for 30 minutes after with the fluorescent intensity was measured on a Tecan M200Pro plate reader and normalized to DAPI.
Cells were plated in 24-well plates with Nunc* Thermanox* Coverslips (Fisher Scientific #12-565-88) at 50,000 cells/cm2 in triplicate. After cells attached overnight, media was replaced with StemPro osteogenesis differentiation media for 21 days, changing every 2 to 3 days. To confirm osteogenesis, cells were stained using Alizarin Red S (ARS) stain solution, prepared with enhanced calcium specificity at a pH of 4.1 to 4.3 as recommended, using ammonium hydroxide. At the end of differentiation step, cells in 24 well-plates were fixed with 4% paraformaldehyde, washed with excess distilled water, stained with Mayer’s haematoxylin for 2 to 5 minutes, rinsed with water, and then stained with 40 mM ARS for 20 min with gentle shaking. The unincorporated dye was washed from the cells four times with excess distilled water while shaking for 5 minutes each wash and cells were cover-slipped with Permount mounting media. Due to temporal changes and reduced osteogenic gene expression at later time points, quantitative reverse transcription–polymerase chain reaction was performed on cells plated in triplicate in 12-well plates at 3000 cells/cm2 and differentiated for 5 days.
Suspended cells at 5 × 105/mL were aliquoted into 10, 15-mL polypropylene conical tubes, with 0.5 mL per tube. Cells were centrifuge at 300 × g at 21°C for 5 min to form a pellet. The tops of the conical tubes remained loose for gas exchange and samples were incubated overnight. The following day, media was replaced with either incomplete differentiation media containing DMEM (Invitrogen # 11054-020), 2 mM L-glutamine (Invitrogen #25030-081), 1X Insulin-Transferrin-Selenium-Plus (BD Biosciences # 354352), 40 μg/mL L-proline (Sigma cat# P5607), 0.1 μM Dexamethasone (Sigma Aldrich, #D4902), and 5μg/mL gentamicin (Invitrogen, #15710-064) or complete differentiation media comprised of incomplete media supplemented with 50 μM L-ascorbic acid 2-phosphate (Invitrogen, #A8960) and 10 ng/mL human transforming growth factor (TGF) beta (R&D Systems, 243-B3-002). Media was replaced every 2 to 3 days for 28 days. During each exchange, pellets were gently agitated to ensure that they would not adhere to the tube wall. At the completion of cell differentiation, two control and chondrocyte pellets were snap frozen in optimal cutting temperature gel, cryosectioned at 5 µm, and stained with Safranin Orange to visualize proteoglycans.
To quantify gene adipo- and osteo- gene expression, ribonucleic acid (RNA) was extracted using an RNeasy mini kit (Qiagen #74104) with DNAse cleanup. Due to the durability and negative charge of cartilage matrix, RNA was extracted from chondrogenic pellets using Trizol extraction and Kimble BioMasherII Closed System Micro Tissue Homogenizer (Fisher, # K7496250010) after which RNA was purified with the RNeasy mini kit as described. cDNA synthesis was performed using 500 ng RNA in 10 µL RNAse free water, random primers (Invitrogen, # 48190011), M-MLVRT (Invitrogen, #28025-013) and appropriate buffers. Relative gene expression for each study subject was determined using the ΔCt method with experimental gene expression compared to glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
The results showed no significant differences existed in cell yield and SVF subpopulation composition within the same patient between harvest procedures 7 to 12 years apart. Further, no change in proliferation rates of cultured ASCs was found, and expanded cells from all patients were capable of tri-lineage differentiation.
By harvesting fat from the same patient at two time points, we have shown that despite the natural human aging process, the prevalence and functional activity of ASCs in an adult mesenchymal stem cell, is highly preserved.
REF: Kokai, L., Traktuev, D., Zhang, L., Merfeld-Clauss, S., DiBernardo, G., Lu, H., Marra, K., Donnenberg, A., Donnenberg, V., Meyer, E., Fodor, P., March, K. and Rubin, J. (2016). Adipose Stem Cell Function Maintained with Age: An Intra-Subject Study of Long-Term Cryopreserved Cells. Aesthetic Surgery Journal, p.sjw197.