Anna Veiga

Disease-corrected haematopoietic progenitors from Fanconi anaemia induced pluripotent stem cells

The generation of induced pluripotent stem (iPS) cells has enabled the derivation of patient-specific pluripotent cells and provided valuable experimental platforms to model human disease. Patient-specific iPS cells are also thought to hold great therapeutic potential, although direct evidence for this is still lacking. Here we show that, on correction of the genetic defect, somatic cells from Fanconi anaemia patients can be reprogrammed to pluripotency to generate patient-specific iPS cells. These cell lines appear indistinguishable from human embryonic stem cells and iPS cells from healthy individuals. Most importantly, we show that corrected Fanconi-anaemia-specific iPS cells can give rise to haematopoietic progenitors of the myeloid and erythroid lineages that are phenotypically normal, that is, disease-free. These data offer proof-of-concept that iPS cell technology can be used for the generation of disease-corrected, patient-specific cells with potential value for cell therapy applications.

Generation of induced pluripotent stem cells from human cord blood using OCT4 and SOX2

Document S1. Supplemental Experimental Procedures and 11 FiguresxDownload (.88 MB ) Document S1. Supplemental Experimental Procedures and 11 FiguresMovie S1. Rhythmically Beating Cardiomyocytes from CBiPS2F-1Specific in vitro differentiation of CBiPS2F-1 into beating cardiomyocytes.xDownload (.75 MB ) Movie S1. Rhythmically Beating Cardiomyocytes from CBiPS2F-1Specific in vitro differentiation of CBiPS2F-1 into beating cardiomyocytes.

New ISSCR Guidelines Underscore Major Principles for Responsible Translational Stem Cell Research

The International Society for Stem Cell Research (ISSCR) task force that developed new Guidelines for the Clinical Translation of Stem Cells discusses core principles that should guide the responsible transition of basic stem cell research into appropriate clinical applications.

Waves of early transcriptional activation and pluripotency program initiation during human preimplantation development.

The events regulating human preimplantation development are still largely unknown owing to a scarcity of material, ethical and legal limitations and a lack of reliable techniques to faithfully amplify the transcriptome of a single cell. Nonetheless, human embryology is gathering renewed interest due to its close relationship with both stem cell biology and epigenetic reprogramming to pluripotency and their importance in regenerative medicine. Carefully timed genome-wide transcript analyses of single oocytes and embryos uncovered a series of successive waves of embryonic transcriptional initiation that start as early as the 2-cell stage. In addition, we identified the hierarchical activation of genes involved in the regulation of pluripotency. Finally, we developed HumER, a database of human preimplantation gene expression, to serve the scientific community. Importantly, our work links early transcription in the human embryo with the correct execution of the pluripotency program later in development and paves the way for the identification of factors to improve epigenetic reprogramming.

Screening for abnormalities of chromosomes X, Y, and 18 and for diploidy in spermatozoa from infertile men participating in an in vitro fertilization-intracytoplasmic sperm injection program

OBJECTIVE To evaluate the frequency of disomy (for chromosomes X, Y, and 18) and of diploidy in the spermatozoa of infertile men undergoing intracytoplasmic sperm injection (ICSI). DESIGN Prospective analysis of sperm nuclei by fluorescence in situ hybridization (FISH). SETTING University-affiliated IVF-ICSI program. PATIENT(S) Semen samples from 19 patients participating in an IVF-ICSI program. INTERVENTION(S) Semen samples were analyzed and prepared for FISH. MAIN OUTCOME MEASURE(S) Semen parameters were evaluated. The frequency of disomy for chromosomes X, Y, and 18 and the frequency of diploidy were analyzed by FISH. RESULT(S) A total of 9,373 spermatozoa from 19 infertile patients were analyzed and compared with spermatozoa from a control group of 5 healthy men. No differences in the frequency of disomy 18 were found, but statistically significant differences in the incidence of sex chromosome disomy and of diploidy were observed. CONCLUSION(S) The study of sperm nuclei by FISH is useful to improve genetic counseling in infertile patients selected for ICSI.

Complete Meiosis from Human Induced Pluripotent Stem Cells

Gamete failure‐derived infertility affects millions of people worldwide; for many patients, gamete donation by unrelated donors is the only available treatment. Embryonic stem cells (ESCs) can differentiate in vitro into germ‐like cells, but they are genetically unrelated to the patient. Using an in vitro protocol that aims at recapitulating development, we have achieved, for the first time, complete differentiation of human induced pluripotent stem cells (hiPSCs) to postmeiotic cells. Unlike previous reports using human ESCs, postmeiotic cells arose without the over‐expression of germline related transcription factors. Moreover, we consistently obtained haploid cells from hiPSCs of different origin (keratinocytes and cord blood), produced with a different number of transcription factors, and of both genetic sexes, suggesting the independence of our approach from the epigenetic memory of the reprogrammed somatic cells. Our work brings us closer to the production of personalized human gametes in vitro. STEM CELLS 2011;29:1186‐1195

Laser blastocyst biopsy for preimplantation diagnosis in the human

A new methodology for blastocyst biopsy that uses a 1.48 microm diode laser is described. Trophectoderm cells are biopsied after laster zona drilling and culture, fixed and processed for fluorescent in situ hybridisation (FISH) analysis. Preliminary results on the efficiency of the procedure and blastocyst recovery rate are promising. Blastocyst laser biopsy is a useful tool in preimplantation genetic diagnosis (PGD) as it allows a more reliable diagnosis and widens the diagnostic possibilities on account of the higher number of cells obtained in the biopsy.

Birth weight and sex ratio after transfer at the blastocyst stage in humans

OBJECTIVE To analyze the birth weights and sex ratio of infants born after blastocyst transfer. DESIGN Retrospective analysis. SETTING Three infertility clinics. PATIENT(S) Patients admitted for IVF with blastocyst transfer. INTERVENTION None. MAIN OUTCOME MEASURE(S) Birth weights and sex ratio of infants born after blastocyst transfer. RESULT(S) Statistically significantly more male infants were born after transfer of fresh blastocysts, either cocultured or cultured in sequential media. No specific differences in birth weight were observed between infants born after blastocyst transfer and those born after spontaneous conception. CONCLUSION(S) More male infants than female infants were born after blastocyst transfer when transfers were performed as soon as the blastocyst stage was reached and male embryos had a faster cleavage rate.

Isolation and Production of Cells Suitable for Human Therapy: Challenges Ahead

Considerable practical hurdles must be overcome prior to the broad application of stem cell therapies. We outline challenges that may vary across different models of cell therapy, including the following broad concepts: issues related to the sourcing of material, and issues related to product manufacturing, shipping, storage and tracking, and standardization.

Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells under Xeno‐free Conditions

The availability of induced pluripotent stem cells (iPSCs) has created extraordinary opportunities for modeling and perhaps treating human disease. However, all reprogramming protocols used to date involve the use of products of animal origin. Here, we set out to develop a protocol to generate and maintain human iPSC that would be entirely devoid of xenobiotics. We first developed a xeno‐free cell culture media that supported the long‐term propagation of human embryonic stem cells (hESCs) to a similar extent as conventional media containing animal origin products or commercially available xeno‐free medium. We also derived primary cultures of human dermal fibroblasts under strict xeno‐free conditions (XF‐HFF), and we show that they can be used as both the cell source for iPSC generation as well as autologous feeder cells to support their growth. We also replaced other reagents of animal origin (trypsin, gelatin, matrigel) with their recombinant equivalents. Finally, we used vesicular stomatitis virus G‐pseudotyped retroviral particles expressing a polycistronic construct encoding Oct4, Sox2, Klf4, and GFP to reprogram XF‐HFF cells under xeno‐free conditions. A total of 10 xeno‐free human iPSC lines were generated, which could be continuously passaged in xeno‐free conditions and maintained characteristics indistinguishable from hESCs, including colony morphology and growth behavior, expression of pluripotency‐associated markers, and pluripotent differentiation ability in vitro and in teratoma assays. Overall, the results presented here demonstrate that human iPSCs can be generated and maintained under strict xeno‐free conditions and provide a path to good manufacturing practice (GMP) applicability that should facilitate the clinical translation of iPSC‐based therapies. STEM CELLS 2010;28:36–44