Abstract 48: Successful Rejuvenation of Mesenchymal Stem Cells From Amputated Limbs of Vascular Patients for Therapeutic Applications
Background: Mesenchymal stem cells (MSC)from patients with critical limb ischemia(CLI) are inferior to healthy donor MSCs (hMSC). Yet cellular therapy may play a role in CLI, and CLI is a relevant setting to test cell rejuvenation techniques. Pooled human platelet lysate (PL) is a novel serum for cell rejuvenation free of xenogens common in fetal bovine serum (FBS).
Objective: To rejuvenate with PL or FBS MSCs cultured from the bone marrow of amputated limbs and compare the biologic function in the setting of CLI (iMSC) or CLI and diabetes (idMSC) to that of hMSC.
Methods: iMSCs (n=4) and idMSCs (n=4) were cultured from the bone marrow of ischemic and ischemic and diabetic amputation specimens and compared to hMSCs (n=4). MSCs were confirmed by FACS and differentiation assays, and doubling time carried out to senescence of comparison groups (passage 9). Secretome analyses (HGF, VEGF, FGFb, MCP-1, IL-6/8) were performed by ELISA after culturing MSCs in basal media + 0.25% HSA. Endothelial cell (EC) proliferation and migration was compared to negative control after stimulation with MSC conditioned media. PKH-stained ECs were co-cultured with these MSCs, and MSC effect on EC invasion quantified under fluorescent imaging. All experiments were performed in quadruplicates with replicates. Statistical analyses were performed with students’ t-test, and ANOVA with Bonferroni or Tukey correction using a significance of P<.05.
Results: MSCs were successfully cultured in both FBS and PL and confirmed by FACS and differentiation assays. Doubling time was delayed in iMSCs and idMSCs compared to hMSCs by passage 7, but PL significantly lessened this effect compared to FBS. Therapeutic levels of HGF, VEGF, and FGFb were identified in the secretome (25-100pg/10,000 MSCs), and conditioned media stimulated EC proliferation over negative control (PBS). EC invasion was significantly stimulated by co-culture with all MSCs compared to ECs alone, and there were no differences between hMSCs and iMSCs or idMSCs on EC proliferation, migration, or invasion.
Conclusion: iMSC and idMSCs rejuvenated in cell culture from early passages (p<7) have similar biologic profiles to that of hMSCs. Streamlined culture rejuvenation paradigms may enable autologous cellular therapy in CLI patients.
Author Disclosures: L. Brewster: None. S. Robinson: None. S. Griffiths: None. A. Rajabalan: None. R. Wang: None. H. Li: None. A. Peister: None. I. Copland: None. T. McDevitt: None.
This research has received full or partial funding support from the American Heart Association.
- © 2014 by American Heart Association, Inc.