Original articleDerivation and characterization of human fetal MSCs: An alternative cell source for large-scale production of cardioprotective microparticles
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 1016–19 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].
Section snippets
Derivation of fetal tissue derived MSCs
The collection of fetal tissue was carried out under a KK Women's and Children's Hospital (KKH) IRB approved protocol (EC200804062) in accordance with guidelines from Singapore Bioethics Advisory Committee [25] which stated that the decision to donate the fetal tissue must be made independently from any decision to abort. Only patients who have already consented to Termination of Pregnancy (TOP) in KKH Outpatient Clinic were recruited. Recruitment was carried out in strict adherence to KKH
Generating MSC cultures from human fetal tissues
We generated five MSC cultures from fetal limb (F1lb, F2lb, F3lb), kidney (F1ki) and liver (F3li) tissues of three fetuses in three independent experiments using feeder- and serum-free culture condition as previously described [21]. A homogenous culture of putative fibroblast-like MSCs migrated out of the tissues and adhered to the plastic culture dish, 2 days after fetal tissues were plated on gelatinized tissue culture plates. This observation was consistent with the defining characteristic
Discussion
The trophic effects of MSCs transplantation on ameliorating the deleterious consequences of myocardial ischemia have been implicated in several studies [10]. Transplantation of MSCs into ischemic myocardium has been shown to induce several tissue responses such as an increased production of angiogenic factors and decreased apoptosis [38] that were better explained by secretion of paracrine factors than by differentiation of MSCs, the so-called paracrine hypothesis.
We have recently demonstrated
Disclosure statement
No potential conflict of interest.
Acknowledgments
We thank J.J. Chee (KKH) for her help in patient recruitment and tissue harvest, and Jayanthi Padmanabhan (BTI) for technical assistance in preparing the secretion.
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