In addition to the lack of

In addition to the lack of a control group, our study has other recognizable limitations. As stated above, this study lacks a fixed/standardized cell dose, making it difficult to establish a real and efficacious cell dosage regime. Characterization of the cellular composition of the resulting SVF in each patient using methods such as flow cytometry would have been optimal, but such equipment was not available at the time of this pilot study. Nevertheless, the cellular composition as well as the secretome of SVF has been largely characterized and described, including “normal” variations in their constitutive percentages (Bourin et al., 2013; da Silva Meirelles et al., 2009; Singer and Caplan, 2011; Yoshimura et al., 2006; Zimmerlin et al., 2010). It is important to note that given the point-of-care nature of the procedure, associated with a short duration for SVF processing and administration, an “in situ” characterization to complement our basic analysis results (cell count, viability, etc.) is difficult, leaving as an option a “confirmatory” analysis of an SVF aliquot performed after the treatment. Future studies, involving more rigorous designs will incorporate such “cell product” characterization.
Conclusions
Author contributions
Declaration of funding interests
Disclosures of potential conflicts of interest
Acknowledgements
Resource Table: , . Resource details The generation of the human iPS cell line, CMT2-FiPS4F1, was carried out using non-integrative Sendai viruses encoding the reprogramming factors OCT3/4, KLF4, SOX2 and cMYC, (Takahashi et al., 2007; Nishimura et al., 2011). For this purpose, fibroblasts from a described patient with caffeic acid phenethyl ester axonal Charcot-Marie-Tooth disease Type 2K ARCMT2K (Sevilla et al., 2008) were obtained from skin biopsies in the CIBERER Biobank. The patient’s fibroblasts presented two mutations in GDAP1 gene (p.Q163X/p.T288NfsX3) in heterozygosis. The presence of these mutations in CMT2-FiPS4F1 cells was confirmed by Sanger sequencing (Fig. 1A). The iPS cell colonies displayed a typical Embryonic Stem (ES) cell-like colony morphology and growth behaviour (Fig. 1B). We confirmed the clearance of the exogenous reprogramming factor genes by quantitative real-time polymerase chain reaction (qPCR) after eight cell culture passages (Fig. 1C). The endogenous expression of the pluripotency-associated transcription factors LEFTY, NANOG and OCT3/4 was evaluated by qPCR (Fig. 1D). Immunofluorescence analysis revealed the expression of the OCT3/4 and NANOG transcription factor proteins, and the surface antigens TRA-1-60 and TRA-1-81, features of pluripotent cells (Fig. 1E). The CMT2-FiPS4F1 cell line was adapted to feeder-free culture conditions without alterations in their karyotype (46, XY) after more than twenty passages (Fig. 1F). Using DNA fingerprinting analysis we confirmed that the CMT2-FiPS4F1 cell line was derived from the patient\'s fibroblasts (Fig. 1G). Finally, we examined the pluripotency of the CMT2-FiPS4F1 cell line by inducing their differentiation through their aggregation in embryoid bodies (EBs), and analysing the expression of markers from the three germ layers (endoderm, mesoderm and ectoderm) by PCR (Fig. 1H) and immunofluorescence (Fig. 1I).
Materials and methods
Resource table. Resource details
Materials and methods
Resource table. Resource details MPB CD34+ cells of a male donor were isolated and differentiated to the megakaryocytic lineage as described before (Heideveld et al., 2015) and FACS sorted to obtain pure CD34+/CD41+ megakaryoblasts. Sorted Megakaryoblasts were transduced with the self-inactivating pRRL.PPT.SF.hOKSMco.GFP.preFRT lentiviral vector (Warlich et al., 2011; Voelkel et al., 2010). Reprogramming was performed on an irradiated mouse embryonic fibroblast (iMEF) feeder layer. The iPSC-like colonies were individually picked 14–20days post-transduction and based on morphology criteria MML-6838-CL2 iPSC was chosen for further examination (Fig. 1A).