Stem Cell Research & Therapy | Full text | A therapy-grade …

Abstract Introduction

Mesenchymal stem cells (MSCs) are a promising source of cells for regenerative therapies. Although they can be isolated easily from several tissues, cell expansion is limited since their properties are lost with successive passages. Hence, pluripotent derived MSCs (PD-MSCs) arise as a suitable alternative for MSC production. Nevertheless, at present, PD-MSC derivation protocols are either expensive or not suitable for clinical purposes.

In this work we present a therapy-grade, inexpensive and simple protocol to derive MSCs from pluripotent stem cells (PSCs) based on the use of platelet lysate (PL) as medium supplement.

We showed that the PD-MSCPL expressed multiple MSC markers, including CD90, CD73, CD105, CD166, and CD271, among others. These cells also show multilineage differentiation ability and immunomodulatory effects on pre-stimulated lymphocytes. Thorough characterization of these cells showed that a PD-MSCPL resembles an umbilical cord (UC) MSC and differs from a PSC in surface marker and extracellular matrix proteins and integrin expression. Moreover, the OCT-4 promoter is re-methylated with mesenchymal differentiation comparable with the methylation levels of UC-MSCs and fibroblasts. Lastly, the use of PL-supplemented medium generates significantly more MSCs than the use of fetal bovine serum.

This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies.

Mesenchymal stem cells (MSC), sometimes also addressed as mesenchymal stromal cells, have been isolated from many different tissues and although some differences may be found according to their origin, most of them share their main features, including multipotent differentiation and immunomodulation [1]. Irrespective of the source of isolation, MSC have been found to be able to modulate the immune response. This feature has been extensively studied in vitro and in vivo in the past years, and MSC are currently assessed in clinical trials for their efficacy in the treatment of many immune-related diseases. Although MSC can be easily isolated from tissues such as bone marrow, umbilical cord or adipose tissue, it has been reported that these cells lose their properties rapidly with time, undergoing cellular senescence [2,3]. Moreover, it is possible that some therapies will require large and repeated doses of MSC. In the case that these therapies involve autologous MSC, there would be some limitations in the number of repeated procedures to obtain the cells. A limitless, economic source of MSC would therefore be a valid alternative when thinking in an autologous, off-the-shelf MSC therapy.

Platelet lysate (PL) is increasingly used instead of fetal bovine serum (FBS) as a medium supplement for growing MSC. PLs advantages have been described extensively, and include its biocompatibility with cell therapy, low cost, and easiness to produce [4,5]. PL contains a very significant amount of growth factors, released by the platelets after lysing in the freeze/thaw cycles [6-8]. These growth factors are involved in many relevant functions in stem cell biology, including basic fibroblast growth factor, insulin-like growth factor and transforming growth factor beta. Moreover, it has been demonstrated that growing MSC in PL-supplemented medium preserves the immunomodulatory ability of the cells [9]. PL supplement has been already used to grow MSC with success, and these cells are used in clinical trials involving MSC without presenting any adverse reaction [10].

Pluripotent stem cells (PSC) can differentiate into any type of adult stem cell. Interestingly, it has been reported that PSC can derive into cells that share many features with MSC isolated from adult tissues, and hence they have been called pluripotent-derived mesenchymal stem cells (PD-MSC) [11-13]. Many papers have described different protocols to derive PD-MSC, and some of them involve some complex manipulations or the use of cell separation methods [14-22]. Even though they are called mesenchymal cells, there are some disagreements between some papers regarding the identity of PD-MSC, and some authors consider that these cells are not related to MSC, based on their gene expression profile [23]. In any case, PD-MSC have been analyzed in many reports and they share many of the features of the adult MSC, including surface markers, multilineage differentiation and immunomodulation. Finally, there are some reports that have analyzed their therapeutic potential, and these cells have been shown to be very potent immunomodulators in animal models [24-27].

We have developed a method to derive PD-MSC using PL as a media supplement (PD-MSCPL). This protocol generates a very significant number of PD-MSC within 3 to 4weeks in a robust and consistent way. We believe that this method can be scaled up at low cost to produce a significant number of PD-MSCPL useful for clinical therapies.

H9 human embryonic stem cells (hES) were purchased from WiCell (Madison, Wisconsin, USA). Induced pluripotent stem cells (iPS) were generated in our laboratory (Maria Questa et al., unpublished observations) by standard techniques. Briefly, foreskin fibroblasts were reprogrammed by transfection with the STEMCCA lentivirus vector, generously obtained from Gustavo Mostovslasky [28]. Several clones have been characterized in our laboratory by demonstrating their pluripotent state and its ability to differentiate into cells from the three germinal layers and by the formation of teratomas. For this paper, we have used both clones FN2.1 and FH. PSC, either hES or iPS, are regularly grown in our laboratory over inactivated (by radiation) mouse embryonic fibroblasts, in Dulbeccos modified Eagles medium (DMEM)/F12 medium supplemented with 10% Knock-Out Serum Replacement, 8ng/ml basic fibroblast growth factor, and penicillinstreptomycin (all from Life Technologies, Carlsbad, California, USA), under standard culture conditions (37C with a 5% carbon dioxide humidified atmosphere). Medium is changed daily. Human umbilical cord mesenchymal stem cells (UC-MSC) were isolated from Wharton jelly tissues. Derivation of UC-MSC was done with due consent from the donors parents. Small pieces of the umbilical cord, excluding the major vessels, were layered onto plastic and cultured in alpha modified Eagles medium supplemented with 10% PL and penicillinstreptomycin. Fibroblasts were obtained from the foreskin of a patient undergoing scheduled surgery, with informed consent from the parents. These cells are grown in DMEM supplemented with 10% FBS and antibiotics. All isolations of primary cells were approved by FLENI Ethical Committee after reviewing the research protocol.

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Stem Cell Research & Therapy | Full text | A therapy-grade ...