A therapy-grade protocol for differentiation of pluripotent stem cells into mesenchymal stem cells using platelet lysate as supplement

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.Fil: Luzzani, Carlos Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; ArgentinaFil: Neiman, Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Garate, Ximena. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Questa, María. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Solari, Claudia María. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Fernandez Espinosa, Darío. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; ArgentinaFil: García, Marcela Nilda. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas; ArgentinaFil: Errecalde, Ana Lía. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas; ArgentinaFil: Guberman, Alejandra Sonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Scassa, María Elida. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; ArgentinaFil: Sevlever, Gustavo. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; ArgentinaFil: Romorini, Leonardo. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; ArgentinaFil: Miriuka, Santiago Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia. Laboratorio de Biología del Desarrollo Celular; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; Argentin

Human Pluripotent Stem Cells and Derived Neuroprogenitors Display Differential Degrees of Susceptibility to BH3 Mimetics ABT-263, WEHI-539 and ABT-199.

Human embryonic stem cells (hESCs) are hypersensitive to genotoxic stress and display lower survival ability relative to their differentiated progeny. Herein, we attempted to investigate the source of this difference by comparing the DNA damage responses triggered by the topoisomerase I inhibitor camptothecin, in hESCs, human induced pluripotent stem cells (hiPSCs) and hESCs-derived neuroprogenitors (NP). We observed that upon camptothecin exposure pluripotent stem cells underwent apoptosis more swiftly and at a higher rate than differentiated cells. However, the cellular response encompassing ataxia-telangiectasia mutated kinase activation and p53 phosphorylation both on serine 15 as well as on serine 46 resulted very similar among the aforementioned cell types. Importantly, we observed that hESCs and hiPSCs express lower levels of the anti-apoptotic protein Bcl-2 than NP. To assess whether Bcl-2 abundance could account for this differential response we treated cells with ABT-263, WEHI-539 and ABT-199, small molecules that preferentially target the BH3-binding pocket of Bcl-xL and/or Bcl-2 and reduce their ability to sequester pro-apoptotic factors. We found that in the absence of stress stimuli, NP exhibited a higher sensitivity to ABT- 263 and WEHI-539 than hESCs and hiPSCs. Conversely, all tested cell types appeared to be highly resistant to the Bcl-2 specific inhibitor, ABT-199. However, in all cases we determined that ABT-263 or WEHI-539 treatment exacerbated camptothecin-induced apoptosis. Importantly, similar responses were observed after siRNA-mediated down-regulation of Bcl-xL or Bcl-2. Taken together, our results suggest that Bcl-xL contrary to Bcl-2 contributes to ensure cell survival and also functions as a primary suppressor of DNA double-strand brake induced apoptosis both in pluripotent and derived NP cells. The emerging knowledge of the relative dependence of pluripotent and progenitor cells on Bcl-2 and Bcl-xL activities may help to predict cellular responses and potentially manipulate these cells for therapeutic purposes in the near future

CPT triggers p53 phosphorylation at Serine 46.

<p>(a) Time course of p53 phosphorylation at serine 46 upon CPT treatment was analyzed by Western blotting using anti-phospho p53 Ser46 (p53pSer46) specific antibody, immediately after (3h), 6h or 15 h post drug removal (3+6 h, 3+15 h, respectively). GAPDH was used as loading control (left panel). p21^Waf1 expression levels upon CPT exposure in hiPSCs and hESCs-derived NP were analyzed by Western blotting immediately after 1μM CPT treatment (3h), 6h or 15h post genotoxic removal (3+6 h, 3+15 h, respectively) using specific antibodies against p21^Waf1 and Actin (right panel) (b) mRNA levels of <i>bcl-2</i>, <i>bax</i> and <i>puma</i> were analyzed by Real Time RT-PCR in CPT treated or untreated hESCs, hiPSCs and NP. GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to undamaged hESCs arbitrarily set as 1. Each bar represents the mean ± SEM of three independent experiments. In all cases, a paired Student's t test was used to compare CPT-treated samples to untreated ones (c) Cell viability was measured by the XTT/PMS assay at 6 h after drug removal in PFT-μ treated or untreated cells (10 μM, 1 h before and during CPT exposure). Results are presented as the percentage of the viability of untreated cells (left panel). PFT-μ treated or untreated cells were harvested 3h after CPT treatment (1 μM over a 3 h period), and DNA oligomers were measured by immunoassay. Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean ± SEM of three independent experiments performed in triplicate (right panel) In all cases, a paired Student's t test was used to compare CPT plus PFT-μ treated samples to CPT treated ones.*P< 0.05, ***P< 0.0001.</p

Pharmacological inhibition or siRNA-mediated down regulation of Bcl-xL sensitize hESCs, hiPSCs and NP to CPT induced DNA damage.

<p>(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were left untreated or treated with increasing concentrations of WEHI-539 (0.1–10 μM) during 15h (top panel) or treated with CPT (1μM) for 3h or pre-treated with WEHI-539 (1μM) for 1h r and then exposed to CPT (bottom panel). Cell viability was measured by the XTT/PMS assay 15 h post WEHI-539 (1μM) addition or 6 h after CPT removal. Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. A paired Student's t test was used to compare WEHI-539-treated samples with untreated ones (top panel) or CPT plus WEHI-539 treated samples to CPT treated ones (bottom panel) *P< 0.05, **P< 0.001. (b) Representative histograms of PI-stained WEHI-539 (1μM) treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells was determined by flow cytometric analysis (c) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP cells were transfected with nt-siRNA or Bcl-xL siRNA (50 nM) and exposed to CPT (1μM for 3h)48 h post-transfection. Representative histograms of PI-stained transfected cells 12 h after genotoxic removal (left panel). mRNA expression levels of <i>bcl-xL</i> in nt-siRNA and Bcl-xL siRNA transfected cell lines were analyzed by Real Time RT-PCR (right panel). GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to nt-siRNA transfectants arbitrarily set as 1. *P < 0.05 **P < 0.001.</p

CPT activates DNA damage response and triggers apoptosis in hiPSCs and hESCs-derived NP.

<p>(a) Immunofluorescence photomicrographs of genotoxic-treated (1μM during 3 h) hiPSCs and NP performed immediately after CPT treatment (1μM during 3 h). The figure shows representative images of cells stained with primary antibodies against ATM phospho-serine1981 (pATM), histoneγH2AX, p53, p53 phospho serine 15 (p53pSer15). Nuclei were counterstained with DAPI. The scale bars represent 100 μm. (b) Cell viability was measured in hESCs, hiPSCs, NP and HF treated or not with 1 μM CPT during 3h by the XTT/PMS assay 6 h after drug removal (top panel). Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. A paired Student’s <i>t</i> test was used to compare CPT treated samples to untreated controls. After a withdrawal period of 3 h CPT-treated cells were harvested, and DNA oligomers were quantified by immunoassay (bottom panel). Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in triplicate. *P< 0.05, **P< 0.001. (c) Time course of caspase-9, caspase-3 and PARP cleavages in hiPSCs and NP upon CPT treatment were analyzed by Western blotting with anti-caspase-9, anti-cleaved-caspase-3 and anti-PARP specific antibodies, immediately after (3 h), 6 h or 15 h post drug removal (3+6, 3+15, respectively). GAPDH was used as loading control.</p

Effects of Bcl-2 selective inhibitor, ABT-199 on the viability of hESCs, hiPSCs NP and HF.

<p>(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were left untreated or treated with increasing concentrations of ABT-199 (0.1–10 μM) during 15 h (top panel) or treated with CPT(1μM) for 3h or pre-treated with ABT-199 (1μM) for 1 h and then treated with CPT (1μM) during 3h (bottom panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-199 addition or 6 h after CPT removal. Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. A paired Student's t test was used to compare ABT-199-treated samples with untreated ones (top panel) or CPT plus ABT-199 treated samples to CPT treated ones (bottom panel) *P< 0.05 (b) Representative histograms of PI- stained ABT-199 treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis (c) NP were transfected with nt-siRNA or Bcl-2 siRNA and exposed to CPT (1μMduring 3h) 48 h post-transfection. Representative pictures showing nt-siRNA or Bcl-2 siRNA transfected NP treated or not with 1μM CPT for 3 h (48 h post-transfection). Images were captured 12 h after genotoxic removal. The scale bars represent 100 μm. Representative histograms of PI-stained transfected cells 12 h after genotoxic removal (left panel). mRNA expression levels of <i>bcl-2</i> in nt-siRNA and Bcl-2siRNA transfected NP were analyzed by Real Time RT-PCR (right panel). GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to nt-siRNA transfectants arbitrarily set as 1. **P < 0.001.</p

hESCs-derived NP and differentiated-neuronal-like cells phenotypic characterization.

<p>(a) Representative images of NP stained with primary antibodies against nestin and DCX (left panel). Representative flow cytometry histograms overlays of H9 hESCs-derived NP are shown to visualize CD133 expression relative to isotype control (right panel) (b) Representative images of NP and differentiated neuronal-like counterparts stained with primary antibodies against MAP-2, MAP-5 and Tuj1. The nuclei were counterstained with DAPI. The scale bars represent 100 μm.</p

ABT-263 reduces cell viability and potentiates CPT-induced apoptosis in hESCs, hiPSCs and NP.

<p>(a) H9 hESCs, FN2.1 hiPSCs, hESCs-derived NP and HF were treated with increasing concentrations of ABT-263 (0.1–1 μM) during 15h (top panel) or pre-treated with ABT-263 (0.1μM) for 1hour and then treated with CPT (1μM during 3h) (middle panel). Cell viability was measured by the XTT/PMS assay 15 h post ABT-263 (0.1μM) addition or 6 h after ABT-263 (0.1μM) and/or genotoxic removal. Results are presented as the percentage of the viability of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in quintuplicate. After 15 h of ABT-263 (0.1μM) addition or a withdrawal period of 3 h ABT-263 (0.1μM) and/or CPT-treated cells were harvested, and DNA oligomers were quantified by immunoassay (bottom panel). Results are presented as the percentage of DNA oligomers of untreated cells. Each bar represents the mean±SEM of three independent experiments performed in triplicate. A paired Student's t test was used to compare ABT-263-treated samples with untreated ones (top panel) or CPT plus ABT-263 treated samples to CPT treated ones (middle and bottom panel)*P< 0.05, **P< 0.001. (b) Representative histograms of PI-stained ABT-263 (0.1μM) treated cells over a 20 h period or untreated unfixed cells. Percentage of PI positive cells (late apoptotic or necrotic) was determined by flow cytometric analysis (c) Caspase-9, caspase-3 activation and PARP cleavage in hESCs, hiPSCs and NP upon CPT (1μM for 3h), ABT-263 (0.1μM for 15 h) or ABT-263 (0.1μM) for 1 h prior and during CPT treatment were analyzed by Western blotting 3 h post genotoxic removal. GAPDH was used as loading control.</p

Bcl-2 family members expression profile in hESCs, hiPSCs, hESCs-derived NP and HF.

<p>(a) mRNA expression levels of <i>bcl-2</i>,<i>bcl-w</i>,<i>bcl-xL</i>,<i>mcl-1</i>,<i>bax</i> and <i>puma</i> were analyzed by Real Time quantitative RT-PCR in hESCs, hiPSCs, NP and HF. GAPDH expression was used as normalizer. Graph shows mRNA fold induction relative to hESCs arbitrarily set as 1. The mean ± S.E. from three independent experiments are shown. In all cases, a paired Student's t test was used to test for significant differences between hESCs and each cell line tested *P < 0.05, ***P< 0.0001. (b) Representative Western blot images are shown. Cellular extracts were prepared and Western blot analyses were carried out using anti-Bcl-2, anti-Bcl-xL and anti-Bax specific antibodies. Actin served as loading control (top panel). Intensities of protein bands corresponding to Bcl-2 and Bcl-xL were measured using computer-assisted densitometric analysis and normalized to the intensity of Actin. Intensities of Bcl-2 and Bcl-xL were also compared to each other and presented as the average ratio for each cell line (bottom panel). A paired Student's t test was performed to test for significant differences between hESCs and each cell line tested *P < 0.05.</p