Fast-proliferating adipose tissue mesenchymal-stromal-like cells for therapy

Abstract

Human mesenchymal stromal cells, whether from the bone marrow or adipose tissue (hASCs), are promising cell therapy agents. However, generation of abundant cells for therapy remains to be a challenge, due to the need of lengthy expansion and the risk of accumulating genomic defects during the process. We show that hASCs can be easily induced to a reversible fast-proliferating phenotype (FP-ASCs) that allows rapid generation of a clinically useful quantity of cells in <2 weeks of culture. Expanded FP-ASCs retain their finite expansion capacity and pluripotent properties. Despite the high proliferation rate, FP-ASCs show genomic stability by array-comparative genomic hybridization, and did not generate tumors when implanted for a long time in an SCID mouse model. Comparative analysis of gene expression patterns revealed a set of genes that can be used to characterize FP-ASCs and distinguish them from hASCs. As potential candidate therapeutic agents, FP-ASCs displayed high vasculogenic capacity in Matrigel assays. Moreover, application of hASCs and FP-ASCs in a fibrin scaffold over a myocardium infarct model in SCID mice showed that both cell types can differentiate to endothelial and myocardium lineages, although FP-ASCs were more potent angiogenesis inducers than hASCs, at promoting myocardium revascularization.

FIG. 1.

Proliferation rate of hASCs is growth medium dependent. (A) The graph describes the proliferation capacity of hASCs in Dulbecco's modified Eagle's medium (DMEM) (open symbols) and in EGM-2 medium (closed symbols) [n=3 for each condition; error bars, standard deviation (SD)]. The red horizontal line marks the 10¹⁰ cell level. Roman numerals (arrows) indicate the times at which β-galactosidase expression was assayed. (B) β-Galactosidase staining (blue), a senescence marker, shows entry of cells in the stationary growth phase. hASCs, human adipose-tissue-derived MSCs. Color images available online at www.liebertpub.com/scd

FIG. 2.

hASCs and FP-ASCs display similar multipotency and immunophenotypes. (A) Panels show microphotographs of FP-ASC and hASC cultures, and their staining with alizarin S and oil red O, following induction to the osteogenic and adipogenic lineages, respectively. (B) The histogram compares the fraction of cells that express the indicated cell surface markers in hASCs (PD 10) from three different patients, with the corresponding early (PD 9) and late (PD 18) passage FP-ASCs. For analysis, CD markers were labeled using the corresponding fluorescence-conjugated antibodies and detected by FACS (n=3, t-Student compared with hASCs for each marker: *P<0.05; ^†P<0.01; error bars, SD). FP-ASCs, fast-proliferating phenotype; PD, population doubling. Color images available online at www.liebertpub.com/scd

FIG. 3.

Safety of FP-ASCs. Luciferase-expressing, early (PD 9) and late (PD 18), FP-ASCs were i.m. implanted in SCID mice and monitored regularly by BLI over a 160-day period. Normal hASCs (PD 10) were also implanted for comparison. (A) The graph represents the luciferase activity recorded from the BLI images of the implantation sites at the indicated days. Data points represent average values±SD (bars) (n=6 for each of the patient-derived cells). (B) Representative image showing a CGH profile from a late-passage FP-ASC patient sample. Shaded areas corresponding to centromeres and constitutive heterochromatin are not considered. The vertical lines, left and right of the CGH profile, represent fluorescence ratio limit values of 0.5–0.75 (red) and 1.0–1.25 (green). CGH ratio values between 1.25 and 0.75 indicate no significant gain or loss of cell DNA relative to control DNA and are considered normal. The actual ratio (black line) between test DNA and control DNA shows no gain or loss of DNA by the chromosomes. Numbers under each diagram indicate chromosome number and the number of chromosomes tested (parenthesis), respectively. SCID, severe-combined immunodeficient mouse; BLI, bioluminescent imaging; CGH, comparative genome hybridization. Color images available online at www.liebertpub.com/scd

FIG. 4.

Vascular capacity of hASCs and FP-ASCs. (A) Representative microphotographs showing tubular structures induced by Matrigel in cultures of hASCs and FP-ASCs, at the indicated times. (B) The histogram represents the quantification of Matrigel-induced tubular structures (circles/mm²). Values for FP-ASCS and hASCs were compared using the t-Student test for each time point (*P<0.01; **P<0.001).

FIG. 5.

In vivo differentiation capacity of heart-implanted hASCs and FP-ASCs. hASCs and FP-ASCs were doubly labeled with either Troponin-1 or PECAM-1 inducible promoter–regulated PLuc-EGFP, and constitutive CMV promoter–regulated RLuc-RFP constructs and seeded in a fibrin patch. The fibrin matrix was then implanted over the ventricular area of myocardium infarct model in mice and imaged by BLI regularly. The ratio of PLuc to RLuc photons (PLuc/RLuc), recorded in the BLI images, was used to evaluate the changes in inducible promoter activity. (A) The histogram represents the change in PLuc/RLuc ratio relative to implantation day (week 0) for each cell type and reporter, n=4. (B) Heart excised after the BLI experiment showing epifluorescent and bioluminescent signals from stem cells seeded in the fibrin patch. (C) Fluorescence confocal microscope images of myocardial sections 3 weeks p.i. showing from left to right: RFP constitutively expressed in all the implanted hASCs and FP-ASCs; EGFP expressed in hASCs and FP-ASCs that differentiated to either the endothelial lineage (PECAM-1 promoter, top panel) or to the cardiomyocytic lineage (Troponin-I promoter, bottom panel); protein antigens, Troponin-I and PECAM-1, detected using the corresponding anti-Troponin-I and anti-PECAM-1 antibodies, shown in gray (top and bottom panels, respectively); overlay of the previous images plus Hoechst staining of nuclei. In each panel, the top row shows FP-ASCs while the bottom row shows hASCs. PLuc, Photinus pyralis luciferase; RLuc, Renilla reniformis luciferase. Color images available online at www.liebertpub.com/scd

FIG. 6.

Vasculogenic effect of therapeutic cells. The histogram shows the fraction (%) of myocardial-vessel area subjacent to the infarct in the MI model subjected to treatment with the fibrin patch, or the fibrin patch plus therapeutic cells. Vascular area was estimated using lectin staining. Values represent the average±SD (*P<0.05; ^†P<0.005, relative to the MI control).