Specific features of ex-obese patients significantly influence the functional cell properties of adipose-derived stromal cells

Abstract

Adipose-derived stromal cells (ADSC) are increasingly used in clinical applications due to their regenerative capabilities. However, ADSC therapies show variable results. This study analysed the effects of specific factors of ex-obese patients on ADSC functions. ADSC were harvested from abdominal tissues (N = 20) after massive weight loss. Patients were grouped according to age, sex, current and maximum body mass index (BMI), BMI difference, weight loss method, smoking and infection at the surgical site. ADSC surface markers, viability, migration, transmigration, sprouting, differentiation potential, cytokine secretion, telomere length and mtDNA copy number were analysed. All ADSC expressed CD73, CD90, CD105, while functional properties differed significantly among patients. A high BMI difference due to massive weight loss was negatively correlated with ADSC proliferation, migration and transmigration, while age, sex or weight loss method had a smaller effect. ADSC from female and younger donors and individuals after weight loss by increase of exercise and diet change had a higher activity. Telomere length, mtDNA copy number, differentiation potential and the secretome did not correlate with patient factors or cell function. Therefore, we suggest that factors such as age, sex, increase of exercise and especially weight loss should be considered for patient selection and planning of regenerative therapies.

Keywords: adipose-derived stromal cells; bariatric surgery; body mass index; cell function; regenerative medicine.

FIGURE 1

(A) Adipose tissue is an abundant source of stem cells. The so‐called adipose‐derived stromal cells (ADSC) can be harvested from patients in large numbers by minimal invasive surgery. They can be cultivated over several passages and cryopreserved. The secretion of growth factors and cytokines and their ability to differentiate into multiple lineages makes them a promising source for regenerative therapies. ADSC can be used in tissue engineering and clinical applications. (B) Spindle‐shaped, plastic adherent ADSC of patient 14 from passage 1. Scale bar 100 μm. (C) Flow cytometric analysis of isolated ADSC (N = 20): positive stem cell markers: CD90, CD73, and CD105; negative markers: Lin (CD34, CD11b, CD19, CD45, HLA‐DR) and CD31. (D) Flow cytometric analysis of isolated ADSC (N = 14): 7AAD stained vital cells and CD142 positive cells. (E): Selection of secreted cytokines and growth factors of isolated ADSC (N = 17) from different patients (displayed in colour)

FIGURE 2

Functional cell assays of isolated ADSC (N = 20). (A) WST‐8/cell viability: Y‐axis shows the percentage increase in absorbance measured after 24 and 72 h for ADSC from different patients. (B) Representative images of ADSC from four different patients from passage 2. Scale bar 100 μm. (C) Migration: Y‐axis shows the number of migrated cells after 48 h per ROI of ADSC from different patients. (D) Representative images of ADSC from four different patients after 48 h, cell nuclei stained with DAPI (blue). Scale bar 200 μm. (E) Transmigration: Y‐axis shows the number of transmigrated cells after 6 h per ROI of ADSC from different patients. (F) Representative images of ADSC from four different patients after 6 h, cell nuclei stained with DAPI (blue). Scale bar 200 μm. (G) Sprouting: Y‐axis shows the cell capacity to sprout after 12 h in a 3D‐fibrin‐matrix of ADSC from different patients. (H) Representative images of cell sprouting from ADSC of four different patients taken after 12 h. Scale bar 200 μm

FIGURE 3

Differentiation assays of isolated ADSC. (A) Adipogenic differentiation: X‐axis shows the percentage of positive stained area after 21 days for ADSC from different patients (N = 17). (B) Representative images of ADSC from four different patients after 21 days stained with Oil red O. Scale bar 500 μm. (C) Osteogenic differentiation: X‐axis shows the percentage of positive stained area after 21 days for ADSC from different patients (N = 13). (D) Representative images of ADSC from four different patients after 21 days stained with alizarin red. Scale bar 2 mm

FIGURE 4

Cell function with regard to patient‐related factors. Y‐axes show the viability, migration, transmigration and sprouting capacities. X‐axes depict the patient‐related factors: (A) age [< 45 years old (n = 10), ≥ 45 years old (n = 10)], (B) sex [female donors (n = 15), male donors (n = 5)] and (C) method of weight loss [bariatric surgery (n = 4), bariatric surgery and change of diet (n = 4), bariatric surgery, change of diet and increase of exercise (n = 3), change of diet and increase of exercise (n = 6)]. *p < 0.05, **p < 0.01. Images were generated with GraphPad Prism (Version 8, GraphPad Software, https://www.graphpad.com)

FIGURE 5

Influence of BMI on functional cell assays. X‐axis shows the viability, migration, transmigration and sprouting of ADSC. Y‐axis depicts: (A) BMI at the time point of ADSC isolation showing no significant correlation, (B) BMI difference due to weight loss with a significant negative correlation between BMI difference and viability, migration and transmigration and (C) maximum BMI that patients had in the past showing no significant correlation. Images were generated with GraphPad Prism (Version 8, GraphPad Software, https://www.graphpad.com)

FIGURE 6

Quantification of telomere length and mtDNA copy number with respect to functional ADSC activity. (A) Y‐axes show the viability, migration, transmigration and sprouting capacities. X‐axes depict the relative telomere length. (B) Y‐axes show the viability, migration, transmigration and sprouting capacities. X‐axes depict the relative mtDNA copy number. (C) Y‐axis shows the relative telomere length and relative mtDNA copy number. X‐axes depict the patient‐related factors: age [<45 years old (n = 10), ≥45 years old (n = 9)], sex [female donors (n = 14), male donors (n = 5)] and method of weight loss [bariatric surgery (n = 4), increase of exercise and change of diet (n = 6), bariatric surgery, change of diet and increase of exercise (n = 3), change of diet and increase of exercise (n = 4)). (D) X‐axes show the relative telomere length and relative mtDNA copy number. Y‐axes depict BMI at the time point of ADSC isolation and BMI difference due to weight loss. Images were generated with GraphPad Prism (Version 8, GraphPad Software, https://www.graphpad.com)