Derivation and characterization of human fetal MSCs: An alternative cell source for large-scale production of cardioprotective microparticles

Abstract

The therapeutic effects of mesenchymal stem cells (MSCs) transplantation are increasingly thought to be mediated by MSC secretion. We have previously demonstrated that human ESC-derived MSCs (hESC-MSCs) produce cardioprotective microparticles in pig model of myocardial ischemia/reperfusion (MI/R) injury. As the safety and availability of clinical grade human ESCs remain a concern, MSCs from fetal tissue sources were evaluated as alternatives. Here we derived five MSC cultures from limb, kidney and liver tissues of three first trimester aborted fetuses and like our previously described hESC-derived MSCs; they were highly expandable and had similar telomerase activities. Each line has the potential to generate at least 10^16–19 cells or 10^7–10 doses of cardioprotective secretion for a pig model of MI/R injury. Unlike previously described fetal MSCs, they did not express pluripotency-associated markers such as Oct4, Nanog or Tra1-60. They displayed a typical MSC surface antigen profile and differentiated into adipocytes, osteocytes and chondrocytes in vitro. Global gene expression analysis by microarray and qRT-PCR revealed a typical MSC gene expression profile that was highly correlated among the five fetal MSC cultures and with that of hESC-MSCs (r² > 0.90). Like hESC-MSCs, they produced secretion that was cardioprotective in a mouse model of MI/R injury. HPLC analysis of the secretion revealed the presence of a population of microparticles with a hydrodynamic radius of 50–65 nm. This purified population of microparticles was cardioprotective at ∼1/10 dosage of the crude secretion.

Introduction

Mesenchymal stem cells (MSCs) are multipotent stem cells that have a limited but robust potential to differentiate into mesenchymal cell types, e.g. adipocytes, chondrocytes and osteocytes, with negligible risk of teratoma formation. MSC transplantation has been used to treat musculoskeletal injuries, improve cardiac function in cardiovascular disease and ameliorate the severity of graft-versus-host-disease [1]. In recent years, MSC transplantations have demonstrated therapeutic efficacy in treating different diseases but the underlying mechanism has been controversial [2], [3], [4], [5], [6], [7], [8], [9]. Some reports have suggested that factors secreted by MSCs [10] were responsible for the therapeutic effect on arteriogenesis [11], stem cell crypt in the intestine [12], ischemic injury [9], [13], [14], [15], [16], [17], [18], and hematopoiesis [19], [20].

We have recently demonstrated that human MSCs derived from human embryonic stem cells (hESC-MSCs) [21] secrete > 200 proteins [22] and that a single bolus administration of hESC-MSCs conditioned medium (CM) 5 min prior to reperfusion significantly reduced infarct size by 60% and improved cardiac function in a pig and mouse model of myocardial ischemia/reperfusion (MI/R) injury [23]. In addition, this cardioprotection was mediated by large complexes of about 50–100 nm in diameter. The size of these large secreted complexes suggests that they are microparticles which are broadly defined as secreted membrane particles in the size range of 0.05–1 μm [24].

A requisite for translating cardioprotective MSC secretion into clinical applications is a clinical grade MSC source but this is currently limited by restricted access to clinical grade hESCs. Therefore, alternative tissue or cell sources that are amenable to the generation of highly expandable, clinical grade MSCs have to be developed. Here we examined fetal tissues as a candidate tissue source.

Five MSC cultures, Fllb, F1ki, F2lb, F3lb and F3li were generated from limb (lb), kidney (ki) and liver (li) tissues of three fetuses in three independent experiments. These fetal MSCs fulfilled the defining criteria of a MSC. They were highly proliferative. Each line has the potential of generating 10^16–19 cells, and therefore the capacity to produce large amount of secretion. More importantly, these cells produce secretion that reduced infarct size in a mouse model of MI/R injury [23]. Like the secretion of hESC-MSCs, this cardioprotection was also mediated by large complexes. Fractionation of the secretion by size exclusion on an HPLC revealed the presence of a population of homogenously sized particles with a hydrodynamic radius of 50–65 nm and these particles were cardioprotective in a mouse model of MI/R injury. The size of these large secreted complexes suggests that they are microparticles which are broadly defined as secreted membrane particles in the size range of 0.05–1 μm [24].