Luzzani et al. Stem Cell Research & Therapy 2015, 6:6 http://stemcellres.com/content/6/1/6 RESEARCH Open Access A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement Carlos Luzzani1*, Gabriel Neiman1, Ximena Garate1, María Questa1, Claudia Solari2, Darío Fernandez Espinosa1, Marcela García3, Ana Lía Errecalde3, Alejandra Guberman2,4, María Elida Scassa1, Gustavo Emilio Sevlever1, Leonardo Romorini1,4 and Santiago Gabriel Miriuka1,3,4* 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. Methods: 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. Results: 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. Conclusions: This protocol can be used to generate a large amount of PD-MSCs with low cost and is compatible with clinical therapies. Introduction for their efficacy in the treatment of many immune- Mesenchymal stem cells (MSC), sometimes also ad- related diseases. Although MSC can be easily isolated dressed as mesenchymal stromal cells, have been iso- from tissues such as bone marrow, umbilical cord or lated from many different tissues – and although some adipose tissue, it has been reported that these cells lose differences may be found according to their origin, most their properties rapidly with time, undergoing cellular of them share their main features, including multipotent senescence [2,3]. Moreover, it is possible that some ther- differentiation and immunomodulation [1]. Irrespective apies will require large and repeated doses of MSC. In of the source of isolation, MSC have been found to be the case that these therapies involve autologous MSC, able to modulate the immune response. This feature has there would be some limitations in the number of repea- been extensively studied in vitro and in vivo in the past ted procedures to obtain the cells. A limitless, economic years, and MSC are currently assessed in clinical trials source of MSC would therefore be a valid alternative when thinking in an autologous, off-the-shelf MSC therapy. * Correspondence: [email protected]; [email protected] Platelet lysate (PL) is increasingly used instead of fetal 1Laboratorio de Biología del Desarrollo Celular, LIAN-Unidad Asociada al bovine serum (FBS) as a medium supplement for grow- CONICET, Fundación FLENI, Ruta 9, Km53, Belen de Escobar, Argentina ing MSC. PL’s advantages have been described exten- 3Cátedra de Citología, Histología y Embriología, Facultad de Ciencias Médicas, Universidad Nacional de La Plata, La Plata, Argentina sively, and include its biocompatibility with cell therapy, Full list of author information is available at the end of the article low cost, and easiness to produce [4,5]. PL contains a © 2015 Luzzani et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Luzzani et al. Stem Cell Research & Therapy 2015, 6:6 Page 2 of 13 http://stemcellres.com/content/6/1/6 very significant amount of growth factors, released by (DMEM)/F12 medium supplemented with 10% Knock-Out the platelets after lysing in the freeze/thaw cycles [6-8]. Serum Replacement, 8 ng/ml basic fibroblast growth factor, These growth factors are involved in many relevant and penicillin–streptomycin (all from Life Technologies, functions in stem cell biology, including basic fibroblast Carlsbad, California, USA), under standard culture condi- growth factor, insulin-like growth factor and transform- tions (37°C with a 5% carbon dioxide humidified atmos- ing growth factor beta. Moreover, it has been demon- phere). Medium is changed daily. Human umbilical cord strated that growing MSC in PL-supplemented medium mesenchymal stem cells (UC-MSC) were isolated from preserves the immunomodulatory ability of the cells [9]. Wharton jelly tissues. Derivation of UC-MSC was done PL supplement has been already used to grow MSC with with due consent from the donor’s parents. Small pieces of success, and these cells are used in clinical trials involv- the umbilical cord, excluding the major vessels, were ing MSC without presenting any adverse reaction [10]. layered onto plastic and cultured in alpha modified Eagle’s Pluripotent stem cells (PSC) can differentiate into any medium supplemented with 10% PL and penicillin– type of adult stem cell. Interestingly, it has been reported streptomycin. Fibroblasts were obtained from the foreskin that PSC can derive into cells that share many features of a patient undergoing scheduled surgery, with informed with MSC isolated from adult tissues, and hence they consent from the parents. These cells are grown in have been called pluripotent-derived mesenchymal stem DMEM supplemented with 10% FBS and antibiotics. All cells (PD-MSC) [11-13]. Many papers have described isolations of primary cells were approved by FLENI Ethical different protocols to derive PD-MSC, and some of them Committee after reviewing the research protocol. involve some complex manipulations or the use of cell separation methods [14-22]. Even though they are called Platelet lysate preparation mesenchymal cells, there are some disagreements bet- PL was produced with some modifications as described ween some papers regarding the identity of PD-MSC, previously [5]. Expired platelet bags were obtained from and some authors consider that these cells are not re- the Hemotherapy Service of FLENI Foundation and fro- lated to MSC, based on their gene expression profile zen at −80°C for at least 24 hours. The units were then [23]. In any case, PD-MSC have been analyzed in many thawed at 37°C, pooled under sterile conditions, and fro- reports and they share many of the features of the adult zen again at −80°C. We prepared several batches, each MSC, including surface markers, multilineage differenti- one containing at least 10 units of platelets obtained ation and immunomodulation. Finally, there are some from at least five different patients. Finally, the pooled reports that have analyzed their therapeutic potential, units were again thawed, aliquoted, and centrifuged at and these cells have been shown to be very potent im- 3,000 × g for 30 minutes. The supernatant was stored munomodulators in animal models [24-27]. at −80°C until use. To use as a supplement, aliquots were We have developed a method to derive PD-MSC using thawed and 2 IU/ml heparin (Sigma-Aldrich, San Luis, PL as a media supplement (PD-MSCPL). This protocol gen- Missouri, USA) were added before medium supplementa- erates a very significant number of PD-MSC within 3 to tion. Alternatively, we modified the protocol described by 4 weeks in a robust and consistent way. We believe that Copland and colleagues for generating fibrinogen- this method can be scaled up at low cost to produce a sig- depleted PL [29]. In this protocol, fibrinogen is excluded nificant number of PD-MSCPL useful for clinical therapies. by adding 10 mM CaCl2 for 1 hour at 37°C. This step gen- erates a dense clot; we then centrifuged the PL at 3,000 × Materials and methods g for 1 hour, collected the supernatant and froze it at −80° Cells and cell pluripotent stem cell culture methods C. We found no difference with either method of PL gen- H9 human embryonic stem cells (hES) were purchased eration regarding the success of PD-MSC differentiation. from WiCell (Madison, Wisconsin, USA). Induced pluripo- However, we prefer this later protocol since it avoids occa- tent stem cells (iPS) were generated in our laboratory sional gelatinization of the medium and produced a sup- (Maria Questa et al., unpublished observations) by standard plement with less debris without affecting results. techniques.Briefly,foreskinfibroblasts were reprogrammed by transfection with the STEMCCA lentivirus vector, gen- Pluripotent-derived mesenchymal stem cell differentiation erously obtained from Gustavo Mostovslasky [28]. Several On day 0 of differentiation, PSC grown either on inactivated clones have been characterized in our laboratory by demon- mouse embryonic fibroblasts,