Adjuvant properties of IFN-γ and GM-CSF in the scFv6.C4 DNA vaccine against CEA-expressing tumors.

Tumor-associated carcinoembryonic antigen (CEA) is a natural target for vaccines against colorectal cancers. Our previous experience with a DNA vaccine with scFv6.C4, a CEA surrogate, showed a CEA-specific immune response with 40% of tumor-free mice after challenge with B16F10-CEA and 47% with MC38-CEA cells. These percentages increased to 63% after using FrC as an adjuvant. To further enhance the vaccine efficacy, we tested GM-CSF and IFNγ as adjuvants. C57BL/6J-CEA2682 mice were immunized 4 times with uP-PS/scFv6.C4, uP-PS/scFv6.C4 + uP-IFNγ, or uP-PS/scFv6.C4 + uP-GMCSF. After one week, the mice were challenged with MC38-CEA, and tumor growth was monitored over 100 days. Immunization with scFv6.C4 and scFv6.C4 + GM-CSF resulted in a gradual increase in the anti-CEA antibody titer, while scFv6.C4 + IFNγ immunization led to a rapid and sustained increase in the titer. The addition of IFNγ also induced higher CD4 + and CD8 + responses. When challenged, almost 80% of the scFv6.C4 + IFNγ-vaccinated mice did not develop tumors, while the others had a significant tumor growth delay. The probability of being tumor-free was 2700% higher using scFv6.C4 + IFNγ than scFv6.C4. The addition of GM-CSF had no additional effect on tumor protection. DNA immunization with scFv6.C4 + IFNγ, but not GM-CSF, increased the antitumor effect via readily sustained specific humoral and cytotoxic responses to CEA.

Effect of GnRH analogues for pituitary suppression on oocyte morphology in repeated ovarian stimulation cycles.

OBJECTIVE: To compare the effect of pituitary suppression regimens on oocyte morphology in consecutive ICSI cycles of the same patients. METHODS: Data was obtained from 200 matched consecutive intracytoplasmic sperm injection (ICSI) cycles performed in 100 couples undergoing the first cycle with the GnRH agonist and the following cycle with the GnRH antagonist regimen, from January 2010 to August 2016, in a private university-affiliated IVF centre. The effects of the pituitary suppression type on oocyte morphology were assessed by multivariate General Linear Models. RESULTS: Mean interval between cycles was 185.32±192.85 days. Maternal age, body mass index, and total FSH dose administered were similar in both patients' cycles. Antagonist cycles presented lower incidence of dark cytoplasm (0.69±3.28% vs. 4.40±17.70%, p=0.047), Smooth endoplasmic reticulum (SER cluster (4.37±11.62% vs. 7.36±17.17%, p=0.046), and ZP defects (6.05±14.76% vs. 11.84±25.13%, p=0.049). Similar numbers of follicles retrieved oocytes, and mature oocytes were observed between the GnRH groups, as well as the fertilisation rate, number of obtained embryos, high-quality embryo rates, and the clinical outcomes. CONCLUSION: GnRH antagonist's inhibitory effect on the ovaries in consecutive ICSI cycles results in improved oocyte maturity and morphology, despite similar laboratory and clinical outcomes, compared to the GnRH agonist treatment.

Immature oocyte incidence: Contributing factors and effects on mature sibling oocytes in intracytoplasmic sperm injection cycles.

OBJECTIVE: The aim of this study was to investigate which factors contribute to the incidence of immature oocytes (germinal vesicle -GV- and metaphase I -MI-) and how they impact the intracytoplasmic sperm injection (ICSI) outcomes of sibling mature oocytes. METHODS: Data from 3,920 cycles performed from June/2010 to August/2016 in a private university-affiliated IVF center were evaluated for the influence of controlled ovarian stimulation protocol (COS) on immature oocytes incidence and its effects on ICSI outcomes. RESULTS: MI (p=0.004) and GV (p=0.029) number were negatively correlated with gonadotropin dose. Patients stimulated by rFSH had increased GV/oocyte rate in both GnRH agonists (p<0.001) and antagonist (p=0.042) protocols, in comparison to rFSH associated with rLH protocol. MI and GV/oocyte rates were negatively correlated to fertilization (p<0.001), high-quality embryo on da p<0.001; GV/oocyte p=0.033) and pregnancy (MI/oocyte p=0.002; GV/oocyte p=0.013) rates. Cycles above a 10.5% MI/oocyte cut-off were correlated to higher response to ovarian stimulation, poor embryo development and almost two times lower pregnancy rate. Immature oocyte incidence is affected by COS and impacts on ICSI outcomes. CONCLUSION: Our evidence suggests that oocytes derived from a cohort with high incidence of maturation fail may have detrimental clinical outcomes.

Sperm DNA fragmentation is correlated with poor embryo development, lower implantation rate, and higher miscarriage rate in reproductive cycles of non-male factor infertility.

OBJECTIVE: To study the implications of sperm DNA fragmentation (SDF) in intracytoplasmic sperm injection cycles for non-male factor infertility. DESIGN: Prospective cohort study. SETTING: Private university-affiliated IVF center. PATIENT(S): Data from 475 cycles performed from June 2016 to June 2017. INTERVENTION(S): Cycles were divided according to SDF rate into two groups: <30% SDF (n = 433) and ≥30% SDF (n = 42). Laboratory and clinical outcomes were compared between groups by generalized linear models adjusted for potential confounders. MAIN OUTCOME MEASURE(S): Embryo quality and miscarriage rates. RESULT(S): Fertilization rate was similar between groups (≥30% SDF, 85.28% ± 1.06% vs. <30% SDF, 90.68% ± 3.61%). Significantly lower rates of normal cleavage speed (≥30% SDF, 61.12% ± 4.21% vs. <30% SDF, 72.53% ± 1.24%), high-quality embryos at day 3 (≥30% SDF, 23.07% ± 5.56% vs. <30% SDF, 36.41% ± 1.53%), blastocyst formation (≥30% SDF, 39.09% ± 2.73% vs. <30% SDF, 58.83% ± 7.59%), blastocyst quality (≥30% SDF, 11.97% ± 1.22% vs. <30% SDF, 30.09% ± 2.39%), and implantation (33.24% ± 1.66% vs. <30% SDF, 46.40% ± 4.61%) were observed in cycles with higher SDF, despite similar pregnancy rates (≥30% SDF, 30.40% vs. <30% SDF, 32.40%). A 2.5-fold miscarriage rate was observed in cycles with an SDF above the established cutoff (≥30% SDF, 42.8% vs. <30% SDF, 16.8%). CONCLUSION(S): Higher SDF is correlated with poor embryo development, lower implantation rate, and higher miscarriage rate in non-male factor infertility intracytoplasmic sperm injection cycles. Since defects in sperm may be hidden, the SDF test can bring additional information to the sperm quality evaluation of men with unknown infertility history.

Serum metabolites as predictive molecular markers of ovarian response to controlled stimulation: a pilot study.

OBJECTIVE: This study aimed to look into the use of serum metabolites as potential biomarkers of response to controlled ovarian stimulation (COS) in patients undergoing intracytoplasmic sperm injection (ICSI) cycles. METHODS: This case-control study analyzed serum samples from 30 patients aged <36 years undergoing COS for ICSI in a university-affiliated assisted reproduction center from January 2017 to August 2017. The samples were split into three groups based on response to COS as follows: poor responders: <4 retrieved oocytes (PR group, n=10); normal responders: ≥ 8 and ≤ 12 retrieved oocytes (NR group, n=10); and hyper-responders: >25 retrieved oocytes (HR, n=10). The metabolic profiles of the serum samples were compared between the groups through Principal Component Analysis (PCA). Receiver Operating Characteristic (ROC) curves were built to assess the power of the model at predicting response to COS. RESULTS: PCA clearly distinguished between PR, NR and HR, and 10 ions were chosen as potential biomarkers of response to COS. These ions were more specific for PR than for NR. The ROC curve considering PR and NR had an area under the curve of 99.6% (95% CI: 88.9 - 100%). CONCLUSION: The preliminary evidence discussed in this study suggests that serum metabolites may be used as predictive molecular markers of ovarian response to controlled stimulation. The integration of clinical and "omics" findings may allow the migration toward an era of personalized treatment in reproductive medicine.

Ovarian response to stimulation and suboptimal endometrial development are associated with adverse perinatal outcomes in intracytoplasmic sperm injection cycles.

OBJECTIVE: To study which factors affect perinatal outcomes in intracytoplasmic sperm injection (ICSI) cycles. METHODS: Data was obtained from 402 live births born to 307 patients undergoing ICSI cycles in a private university-affiliated IVF center between Jan/2014 and Dec/2015. The influences of the cycles' characteristics on the number of gestational weeks to livebirth (GW), baby birth weight (BW), and baby birth length (BL) were evaluated by linear regression models, adjusted for maternal age and body mass index, number of transferred embryos, number of gestational sacs, and number of born infants. In a subsequent analysis, GW, BW and baby sex were utilized for cycle classification into the groups Appropriate for gestational age (AGA n=256) and Small for gestational age (SGA n=146), which were compared by general linear models adjusted for the same confounder variables. RESULTS: The number of follicles (ß=-0.069 p=0.018) and retrieved oocytes (ß=-0.087 p=0.049) were negatively correlated with BL. The endometrial thickness was positively correlated with GW (ß=0.198 p=0.003) and BW (ß=28.351 p=0.044). When each baby was classified into AGA and SGA groups, it was observed that SGA babies were derived from cycles with higher estradiol levels at hCG day (SGA: 3897.01±550.35 vs. AGA: 2324.78±101.86 p=0.006) and higher number of retrieved oocytes (SGA: 16.70±1.78 vs. AGA: 12.92±0.42 p=0.042). The endometrial thickness was significantly lower in the SGA group (SGA: 10.2±0.23 vs. AGA: 11.68±0.17 vs. p=0.029). CONCLUSION: Higher ovarian response to stimulation and suboptimal endometrial development are associated with adverse perinatal outcomes in ICSI cycles.

Predictive factors for biochemical pregnancy in intracytoplasmic sperm injection cycles.

The aim of this study was to investigate which factors contribute to the incidence of biochemical pregnancy (BP) in intracytoplasmic sperm injection (ICSI) cycles. This cohort study included cycles performed from June 2010 to September 2016 in a private, university-affiliated IVF centre. Cycles were split into four groups, depending on the pregnancy outcomes: Clinical Pregnancy (CP, n = 903), Biochemical Pregnancy (BP, n = 55), Miscarriage (MI, n = 142) and Negative Pregnancy (NP, n = 2034). The effects of ovarian stimulation, laboratory data and seminal parameters on pregnancy outcomes were evaluated using adjusted general linear models. Discriminant analyses were conducted to construct a model for pregnancy prediction and to establish cut-offs for BP. The total sperm count (p = 0.035), total and progressive sperm motility (p = 0.001 and p = 0.023, respectively), total motile sperm count (TMSC, p = 0.029) and the endometrial thickness (p < 0.001) were lower among BP group cycles. Lower rates of high-quality cleavage-stage embryos were observed in the BP group compared to CP and MI groups (p < 0.001). In discriminant analyses, cut-offs for BP prediction were established for the following factors: endometrial thickness < 11 mm, sperm motility < 55.5% and total dose of follicle-stimulating hormone (FSH)> 2400 IU. The incidence of biochemical pregnancy was four times higher when the aforementioned factors did not meet the defined cut-offs. The combination of suboptimal endometrial development and poor seminal and embryo quality contribute to an increased incidence of biochemical pregnancy in ICSI cycles.

scFv6.C4 DNA vaccine with fragment C of Tetanus toxin increases protective immunity against CEA-expressing tumor.

The carcinoembryonic antigen (CEA) is the main tumor-associated antigen of colorectal cancers. Previously, we developed a DNA vaccine using scFv6.C4, a CEA surrogate, against CEA-expressing tumors; 40% of the vaccinated mice were tumor-free after tumor challenge. In order to enhance vaccine efficacy, fragment C of Tetanus Toxin (FrC) was tested as adjuvant. C57BL/6J-CEA2682 mice were electroporated intramuscularly 4 times with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4, challenged by s.c. injection of 1 × 105 MC38-CEA cells, and tumor growth was monitored over 100 days. The humoral and cellular immune responses were assessed by ELISA, immunocytochemistry, in-vitro lymphocyte proliferation, and CTL cytotoxicity assays. Immunization with uP-PS/scFv6.C4-FrC or uP-PS/scFv6.C4 induced similar anti-CEA antibody titers. However, immunocytochemistry analysis showed stronger staining with uP-PS/scFv6.C4-FrC-immunized mice sera. When challenged with MC38-CEA cells, 63% of the FrC-vaccinated mice did not develop tumors, half of the rest had a significant tumor growth delay, and the probability of being free of tumors was on average 40% higher than that of scFv6.C4-immunized mice. Addition of the adjuvant led to higher CD4+ and CD8+ proliferative responses and strong CD8+ CTL response against MC38-CEA cells. DNA immunization with scFv6.C4 and FrC increased antitumor effect via induction of high and specific humoral and cellular immune responses to CEA.

Preimplantation genetic testing for monogenic diseases: a Brazilian IVF centre experience.

OBJECTIVE: To describe the cases of preimplantation genetic testing for monogenic diseases (PGT-M) in fertile couples who had undergone intracytoplasmic sperm injection (ICSI) cycles in a Brazilian in vitro fertilisation (IVF) centre and determine whether these cases were different from those reported from the European Society of Human Reproduction and Embryology (ESHRE). METHODS: This retrospective collection included data obtained from ICSI-PGT-M cycles between 2011 and 2016. The disease indication, number of biopsied embryos, biopsy stage, diagnosed and affected embryos, and cycles with embryo to transfer as well as implantation, pregnancy and miscarriage rates were analysed and compared to cycles without genetic diagnosis (PGT) and with ESHRE PGD Consortium collection XIV-XV. RESULTS: From 5,070 cycles performed, 72 had indications for PGT-M. The most common time for biopsy was cleavage-stage; 93% of the embryos had a diagnostic result, 59.4% of which were genetically transferable, resulting in 68% of the cycles with transferred embryos, a 22.1% implantation rate, and a 28.6% pregnancy rate. No differences in clinical outcomes of cycles with PGT-M or without PGT were observed. The day of biopsy and diagnostic success as well as implantation, pregnancy and miscarriage rates were similar to ESHRE collection. CONCLUSIONS: Although the proportion of cases with PGT-M was low, its efficacy was similar to what was reported in the European collection and represents a viable alternative for families at risk of transmitting a genetic disorder to their offspring. The main difference between our and ESHRE collection were the disease indications, which reflected the admixed, multi-ethnic Brazilian population.

Role of religion, spirituality, and faith in assisted reproduction.

Purpose: The purpose of this study is to evaluate the impact of the patient's faith, religion, and spirituality on the outcomes of intracytoplasmic sperm injection (ICSI) cycles. Materials and methods: Eight hundred and seventy-seven patients received a questionnaire containing information on faith, religiosity, and spirituality and the results of the questionnaires were correlated with ICSI outcomes. Patients stated to be Catholic (n = 476), spiritists (n = 93), Evangelical (n = 118), and other religion (n = 32), and 78 did not identify with any religious group. Results: A significant increase in fertilization, high-quality embryos, and pregnancy rate was found among Spiritists and Evangelicals. Patients who included the infertility diagnosis and treatment in their prayers showed an increased pregnancy rate, and those who reported their faith to be affected by the infertility diagnosis presented a decreased high-quality embryos rate. The high-quality embryos rate was increased among patients who answered that their faith contributed to their decision to undergo infertility treatment. The cycle's cancelation was negatively correlated with the frequency of religious meetings, and the frequency of prayers was positively correlated with the response to ovarian stimulation. Finally, belief in treatment success positively influenced the embryo quality. Conclusion: The findings suggest that spirituality plays a role in adjusting the psychological aspects of an infertile patient.

Lipidomic profile as a noninvasive tool to predict endometrial receptivity.

For the present study we asked whether the endometrial fluid lipidomic may be a useful approach to predict endometrial receptivity in freeze-all cycles. For this case-control study, endometrial fluid samples were collected from 41 patients undergoing freeze-all cycles. Samples were split depending on the pregnancy outcome: positive group (n = 24) and negative group (n = 17). Data were acquired by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were applied. A list of potential biomarker ion ratios was obtained and the values were used to build a receiver operating characteristic (ROC) curve to predict pregnancy success. The lipid categories were attributed by LIPID MAPS database. Ion ratios were established according to their correlations and used for the analysis. The PCA showed a tendency of separation between the studied groups, whereas the PLS-DA was able to clearly distinguish them. Fifteen ratios (13 hyper-represented in the negative and two hyper-represented in the positive group) were selected according to their importance for model prediction. These ratios were used to build the ROC curve, which presented an area under curve of 84.0% (95%CI: 69.2-97.4%; p = 0.009). These findings suggest that lipidomic profiling of endometrial fluid may be a valuable tool for identifying the time interval comprising the window of implantation.

Overcoming male factor infertility with intracytoplasmic sperm injection.

OBJECTIVE: To evaluate the effect of male factor infertility on intracytoplasmic sperm injection (ICSI) outcomes compared with a control group presenting isolated tubal factor. METHOD: This retrospective study included 743 couples undergoing ICSI as a result of isolated male factor and a control group consisting of 179 couples undergoing ICSI as a result of isolated tubal factor, performed in a private university- -affiliated in vitro fertilization center, between January/2010 and December/2016. Patients were divided into two groups according to maternal age: women ≤35 years old and >35 years old. The effects of infertility causes on laboratorial and clinical ICSI outcomes were evaluated using Student's t-test and (2 test. RESULTS: No differences in controlled ovarian stimulation outcomes were observed between male factor cycles and tubal factor cycles in the two age groups. Implantation (male factor 35.5% vs. tubal factor 32.0%, p=0.340), pregnancy (male factor 46.9% vs. tubal factor 40.9%, p=0.184) and miscarriage (male factor 10.3% vs. tubal factor 10.6%, p=0.572) rates were similar between the infertility groups, irrespective of female age. Considering maternal age, the cancelation rate was higher in older women (>35 years old) undergoing ICSI as a result of male factor infertility (17.4% vs. 8.9%, p=0.013). CONCLUSION: Our results showed that there is no difference in the outcomes of pregnancy between couples with male or tubal factor infertility, which indicates that ICSI surpasses the worse specific outcomes associated with male factor.

Zika Virus Outbreak - Should assisted reproduction patients avoid pregnancy?

OBJECTIVE: To discuss the requirement from the National Health Surveillance Agency (ANVISA), for assisted reproduction treatment patients to undergo laboratory tests for ZIKV detection, and if the public health authorities and government leaders' recommendations to women simply avoid pregnancy is prudent. METHODS: This study was performed in a university-affiliated in vitro fertilization center in Brazil. We present a critical discussion on the risk of microcephaly due to ZIKV infection and the prevalence of other harmful pathogens to vulnerable pregnant women and infants. We assessed, 954 patients undergoing intracytoplasmic sperm injection cycles (ICSI), between April and November of 2016, concerning the results of ZIKV test, according to different regions in Brazil. RESULTS: Patients undergoing ICSI cycles were split into groups, according to their region of origin: 28 (3.0%) were from the North, 27 (2.8%) were from the Northeast, 40 (4.2%) were from the Midwest, 830 (87.2%) were from the Southeast, and 29 (3.0%) were from the South. Concerning the diagnosis, 112 samples had a positive or inconclusive result for ZIKV, by chromatography immunoassay. These samples were re-analyzed by ELISA and no result was positive. All positive results were from the Southeast region and none from the Northeast or Midwest regions, which are considered endemic regions. CONCLUSION: ZIKV test before the onset of assisted reproduction treatments does not rule out the risk of the infection during pregnancy. In addition, although ZIKV infection risk is extremely high, the microcephaly risk due to ZIKV is not higher than the risk of miscarriage and birth defects due to other recognized pathogens.

Overcoming male factor infertility with intracytoplasmic sperm injection / Superando o fator masculino de infertilidade com injeção intracitoplasmática de espermatozoides

Summary Objective: To evaluate the effect of male factor infertility on intracytoplasmic sperm injection (ICSI) outcomes compared with a control group presenting isolated tubal factor. Method: This retrospective study included 743 couples undergoing ICSI as a result of isolated male factor and a control group consisting of 179 couples undergoing ICSI as a result of isolated tubal factor, performed in a private university- -affiliated in vitro fertilization center, between January/2010 and December/2016. Patients were divided into two groups according to maternal age: women ≤35 years old and >35 years old. The effects of infertility causes on laboratorial and clinical ICSI outcomes were evaluated using Student's t-test and (2 test. Results: No differences in controlled ovarian stimulation outcomes were observed between male factor cycles and tubal factor cycles in the two age groups. Implantation (male factor 35.5% vs. tubal factor 32.0%, p=0.340), pregnancy (male factor 46.9% vs. tubal factor 40.9%, p=0.184) and miscarriage (male factor 10.3% vs. tubal factor 10.6%, p=0.572) rates were similar between the infertility groups, irrespective of female age. Considering maternal age, the cancelation rate was higher in older women (>35 years old) undergoing ICSI as a result of male factor infertility (17.4% vs. 8.9%, p=0.013). Conclusion: Our results showed that there is no difference in the outcomes of pregnancy between couples with male or tubal factor infertility, which indicates that ICSI surpasses the worse specific outcomes associated with male factor.

Resumo Objetivo: Avaliar o efeito do fator masculino de infertilidade em resultados de injeção intracitoplasmática de espermatozoides (ICSI) em comparação com um grupo controle que apresenta o fator tubário isolado. Método: Este estudo retrospectivo incluiu 743 casais submetidos a ICSI por fator masculino e 179 casais por fator tubário, realizada em um centro privado de fertilização in vitro associado à universidade, entre janeiro de 2010 e dezembro de 2016. Os pacientes foram divididos em dois grupos de acordo com a idade materna: mulheres ≤ 35 e > 35 anos de idade. Os efeitos das causas de infertilidade nos resultados laboratoriais e clínicos da ICSI foram avaliados pelos testes T de Student e Qui-quadrado. Resultados: Não foram observadas diferenças nos parâmetros de estimulação ovariana entre os ciclos com fatores masculinos e com fatores tubários. A taxa de implantação (fator masculino 35,5% vs. fator tubário 32,0%, p=0,340), de gravidez (fator masculino 46,9% vs. fator tubário 40,9%, p=0,184) e de aborto (fator masculino 10,3% vs. fator tubário 10,6%, p=0.572) foram semelhantes entre os grupos de infertilidade, independentemente da idade feminina. Considerando a idade materna, a taxa de cancelamento foi maior em mulheres > 35 anos cuja causa de infertilidade era o fator masculino (17,4% vs. 8,9%, p=0,013). Conclusão: Não há diferenças nos resultados de gravidez entre casais com infertilidade dos fatores masculino ou tubário isolados, o que indica que ICSI supera os piores resultados associados ao fator masculino.

Trypanosoma cruzi extracellular amastigotes trigger the protein kinase D1-cortactin-actin pathway during cell invasion.

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

The Anti-Tumor Effects of Adipose Tissue Mesenchymal Stem Cell Transduced with HSV-Tk Gene on U-87-Driven Brain Tumor.

Glioblastoma (GBM) is an infiltrative tumor that is difficult to eradicate. Treating GBM with mesenchymal stem cells (MSCs) that have been modified with the HSV-Tk suicide gene has brought significant advances mainly because MSCs are chemoattracted to GBM and kill tumor cells via a bystander effect. To use this strategy, abundantly present adipose-tissue-derived mesenchymal stem cells (AT-MSCs) were evaluated for the treatment of GBM in mice. AT-MSCs were prepared using a mechanical protocol to avoid contamination with animal protein and transduced with HSV-Tk via a lentiviral vector. The U-87 glioblastoma cells cultured with AT-MSC-HSV-Tk died in the presence of 25 or 50 µM ganciclovir (GCV). U-87 glioblastoma cells injected into the brains of nude mice generated tumors larger than 3.5 mm2 after 4 weeks, but the injection of AT-MSC-HSV-Tk cells one week after the U-87 injection, combined with GCV treatment, drastically reduced tumors to smaller than 0.5 mm2. Immunohistochemical analysis of the tumors showed the presence of AT-MSC-HSV-Tk cells only within the tumor and its vicinity, but not in other areas of the brain, showing chemoattraction between them. The abundance of AT-MSCs and the easier to obtain them mechanically are strong advantages when compared to using MSCs from other tissues.

Identification, selection, and enrichment of cardiomyocyte precursors.

The large-scale production of cardiomyocytes is a key step in the development of cell therapy and tissue engineering to treat cardiovascular diseases, particularly those caused by ischemia. The main objective of this study was to establish a procedure for the efficient production of cardiomyocytes by reprogramming mesenchymal stem cells from adipose tissue. First, lentiviral vectors expressing neoR and GFP under the control of promoters expressed specifically during cardiomyogenesis were constructed to monitor cell reprogramming into precardiomyocytes and to select cells for amplification and characterization. Cellular reprogramming was performed using 5'-azacytidine followed by electroporation with plasmid pOKS2a, which expressed Oct4, Sox2, and Klf4. Under these conditions, GFP expression began only after transfection with pOKS2a, and less than 0.015% of cells were GFP(+). These GFP(+) cells were selected for G418 resistance to find molecular markers of cardiomyocytes by RT-PCR and immunocytochemistry. Both genetic and protein markers of cardiomyocytes were present in the selected cells, with some variations among them. Cell doubling time did not change after selection. Together, these results indicate that enrichment with vectors expressing GFP and neoR under cardiomyocyte-specific promoters can produce large numbers of cardiomyocyte precursors (CMPs), which can then be differentiated terminally for cell therapy and tissue engineering.