Mona Sheikhi

Clonal culturing of human embryonic stem cells on laminin-521/E-cadherin matrix in defined and xeno-free environment

Lack of robust methods for establishment and expansion of pluripotent human embryonic stem (hES) cells still hampers development of cell therapy. Laminins (LN) are a family of highly cell-type specific basement membrane proteins important for cell adhesion, differentiation, migration and phenotype stability. Here we produce and isolate a human recombinant LN-521 isoform and develop a cell culture matrix containing LN-521 and E-cadherin, which both localize to stem cell niches in vivo. This matrix allows clonal derivation, clonal survival and long-term self-renewal of hES cells under completely chemically defined and xeno-free conditions without ROCK inhibitors. Neither LN-521 nor E-cadherin alone enable clonal survival of hES cells. The LN-521/E-cadherin matrix allows hES cell line derivation from blastocyst inner cell mass and single blastomere cells without a need to destroy the embryo. This method can facilitate the generation of hES cell lines for development of different cell types for regenerative medicine purposes.

Clinical grade vitrification of human ovarian tissue: an ultrastructural analysis of follicles and stroma in vitrified tissue

BACKGROUND Cancer therapy is one of many conditions which may diminish the ovarian reserve. Banking of human ovarian tissue has become an option for the preservation of female fertility. We have shown that vitrification is an excellent method to cryopreserve ovarian tissue. To carry out vitrification in a clinical setting, we have developed a clinical grade closed system to avoid direct contact of ovarian tissue with liquid nitrogen. METHODS Ovarian tissue was obtained by biopsy from 12 consenting women undergoing Caesarean section. Tissues were vitrified in cryotubes, using dimethyl sulphoxide, 1,2-propanediol, ethylene glycol and polyvinylpyrrolidon as cryoprotectants. Non-vitrified and warmed-vitrified tissue was compared by light and electron microscopic morphology of the follicles within the tissues. RESULTS We did not see any differences in the light or electron microscopic ultrastructure of oocytes between non-vitrified and vitrified tissues. No irreversible subcellular alterations in vitrified tissues were seen. CONCLUSIONS The ultrastructure of follicles within the vitrified human ovarian tissue was well preserved using cryotube in a closed vitrification system to avoid direct contact of liquid nitrogen. The system is compatible with the European tissue directive.

Adult human and mouse ovaries lack DDX4-expressing functional oogonial stem cells.

The generally accepted viewpoint for more than 50 years has been that the number of oocytes is fixed in fetal or neonatal ovaries, and therefore, oocytes cannot renew themselves in postnatal or adult life. Over the past decade, however, the traditional viewpoint has been challenged by a number of investigators who have presented evidence that postnatal follicular renewal occurs in mammals, and that mitotically active oogonial stem cells (OSCs) exist in postnatal mouse ovaries. Health, Inc. All rights reserved. 30 Obstetrical and Gynecological Survey This letter to the editor presents experimental evidence that disputes the existence of mitotically active OSC in postnatal mouse ovaries. The results presented here are the summary of research conducted independently in 4 laboratories. A previous study (White et al.Nat Med. 2012;18:413–421) reported that OSCs could be purified from adult human and mouse ovaries by use of DEAD box polypeptide 4 (DDX4) antibody–based fluorescence-activated cell sorting (FACS), and that after in vitro manipulation, these isolated OSCs could form oocytes. Based on the well-established cytoplasmic location of DDX4, the use of this protein as a cell surface marker is controversial. Using the same DDX4 antibody–based FACS approach as in the White et al study, the investigators isolated a population of cells from human ovarian cortical tissue biopsied from 16 fertile reproductive-age women who had had at least 1 previous live birth. No DDX4 messenger RNA (mRNA) expression was detected using quantitative polymerase chain reaction in these cells or by a more sensitive single-cell mRNA sequencing analysis that could detect low expression of DDX4. In additional experiments, the sorted human ovarian cells were cultured as described in the previous study. Although no DDX4 expression was detected in the cultured DDX4-positive cells (cultured-POS) or cultured DDX4-negative cells (cultured-NEG) by immunofluorescence staining, the cultured-POS cells and cultured-NEG cells both bound tightly to the DDX4-specific antibody in FACS and became DDX4-positive after culture. The previous study had reported that oocytes enclosed in follicles regenerated 1 week after the DDX4-positive human OSCs were injected into human ovarian cortical tissues that were subsequently xenografted into female severe combined immunodeficient mice. The investigators repeated this experiment and labeled the cultured-POS cells with stable enhanced green fluorescent protein (EGFP) expression. After culturing and expanding this cell population, EGFP-expressing cultured-POS cells were injected into human ovarian cortical tissue biopsies, and these cortical tissues were then xenografted into female severe combined immunodeficient mice for further growth. Grafts were analyzed 1 week, 2 weeks, and 4 weeks after transplantation of the EGFP–cultured-POS cells into the human cortical tissues. The results of this experiment showed that EGFP-positive cells could be observed in the vicinity of the injection sites, but the absence of any EGFP-positive oocytes demonstrated that the DDX4-positive human cells obtained with the DDX4 antibody are not functional stem cells and cannot regenerate oocytes. To confirm these findings that the DDX4-specific antibody–based FACS does not select for a specific cell population expressing DDX4, the same FACS was performed with mouse cells from several organs (including adult liver, spleen, and kidney) that do not express DDX4. DDX4-positive cell populations were obtained from cells of these organs, which provide additional evidence that use of the DDX4-specific antibody using the FACS protocol (critical in purifying the reported OSCs) does not select for DDX4-expressing cells. These findings provide evidence that supports the traditional view that no postnatal follicular renewal occurs in mammals, and no mitotically active DDX4-expressing female germline progenitors exist in postnatal mouse ovaries.

Novel PRD-like homeodomain transcription factors and retrotransposon elements in early human development

Transcriptional program that drives human preimplantation development is largely unknown. Here, by using single-cell RNA sequencing of 348 oocytes, zygotes and single blastomeres from 2- to 3-day-old embryos, we provide a detailed analysis of the human preimplantation transcriptome. By quantifying transcript far 5′-ends (TFEs), we include in our analysis transcripts that derive from alternative promoters. We show that 32 and 129 genes are transcribed during the transition from oocyte to four-cell stage and from four- to eight-cell stage, respectively. A number of identified transcripts originates from previously unannotated genes that include the PRD-like homeobox genes ARGFX, CPHX1, CPHX2, DPRX, DUXA, DUXB and LEUTX. Employing de novo promoter motif extraction on sequences surrounding TFEs, we identify significantly enriched gene regulatory motifs that often overlap with Alu elements. Our high-resolution analysis of the human transcriptome during preimplantation development may have important implications on future studies of human pluripotent stem cells and cell reprograming.

Full‐term newborn after repeated ovarian tissue transplants in a patient treated for Ewing sarcoma by sterilizing pelvic irradiation and chemotherapy

We report the first successful transplantation of cryopreserved ovarian cortical tissue into heavily irradiated tissues in a patient who had received sterilizing pelvic radiotherapy (54 Gy) and 40 weeks of intensive high‐dose chemotherapy for the treatment of Ewings sarcoma 14 years earlier. Repeated transplantation procedures were required to obtain fully functional follicular development. Enlargement of the transplants over time and increase of the size of the uterus were demonstrated on sequential ultrasonographic examinations. Eggs of good quality that could be fertilized in vitro were obtained only after a substantial incremental increase of the amount of ovarian tissue transplanted. Single embryo replacement resulted in a normal pregnancy and the birth of a healthy child by cesarean section at full‐term. No neonatal or maternal postoperative complications occurred. Women facing high‐dose pelvic radiotherapy should not be systematically excluded from fertility preservation options, as is currently the trend.

Apoptosis of human ovarian tissue is not increased by either vitrification or rapid cooling

This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in vitrified and rapidly cooled human ovarian tissue. Apoptosis was assessed 30 min and 24h after warming using transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation, as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryoprotectant solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis and either protocol could be an alternative to slow cooling of ovarian tissue. This study evaluated the incidence of morphological changes, as assessed by light microscopy, and apoptosis in human ovarian tissue cryopreserved using two different methods, i.e. vitrification and rapid cooling. Apoptosis was assessed in tissue 30 min and 24h after warming using transmission electron microscopy and terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) assay and DNA fragmentation as determined by gel electrophoresis. The results showed no significant changes in morphology, chromatin condensation, DNA fragmentation or TUNEL-positive cells in follicles attributable to cryopreservation or exposure to the cryopreservation solutions alone. In conclusion, the cryopreservation protocols did not affect the incidence of apoptosis in human ovarian tissue and either protocol could be an alternative to slow cooling for the preservation of ovarian tissue.

Preservation of human ovarian follicles within tissue frozen by vitrification in a xeno-free closed system using only ethylene glycol as a permeating cryoprotectant

OBJECTIVE To study the preservation of follicles within ovarian tissue vitrified using only one or a combination of three permeating cryoprotectants. DESIGN Experimental study. SETTING University hospital. DONOR(S) Ovarian tissue was donated by consenting women undergoing elective cesarean section. INTERVENTION(S) Ovarian tissue was vitrified in closed sealed vials using either a combination of dimethyl sulfoxide, 1,2-propanediol, and ethylene glycol (EG), or only EG as permeating cryoprotectants. MAIN OUTCOME MEASURE(S) Ovarian tissue was vitrified with the use of two vitrification methods. Tissue from the same donor was used for comparison of two different solutions. The morphology of the follicles was evaluated after vitrification, warming, and culture by light microscopy and transmission electron microscopy. Apoptosis was assessed by immunohistochemistry for active caspase-3 in fresh and vitrified tissue. RESULT(S) Light and electron microscopic analysis showed equally well preserved morphology of oocytes, granulosa cells, and ovarian stroma when either of the vitrification solutions was used. No apoptosis was observed in primordial and primary follicles. CONCLUSION(S) Using only EG as a permeating cryoprotectant in a closed tube gives as good ultrastructural preservation of ovarian follicles as a more complicated system using several cryoprotectants.

Effect of Previous Chemotherapy on the Quality of Cryopreserved Human Ovarian Tissue In Vitro

Background Cryopreservation of ovarian tissue has been widely accepted as an option for fertility preservation among cancer patients. Some patients are exposed to chemotherapy prior to ovarian tissue cryopreservation. Consequently, assessment of the developmental capacity of human ovarian tissue after chemotherapy is of primary importance. Materials In order to study the impact of previous chemotherapy on in vitro development and viability of ovarian follicles, quality control samples from 34 female cancer patients at median age of 15 years (range 1‒35), cryopreserved for fertility preservation before (n = 14) or after (n = 20) initiation of chemotherapy, were thawed and cultured for 7 days. The morphology and developmental stages of ovarian follicles were studied by light microscopy before and after culture. Possible associations between follicular densities, age and exposure to alkylating agents, expressed as cyclophosphamide equivalent dose (CED) were tested. Results Exposure to chemotherapy significantly impaired the survival and development of ovarian follicles in culture. After seven days, significantly higher densities of intermediary, primary and secondary follicles and lower densities of atretic follicles was detected in the samples collected before chemotherapy. Increasing dose of alkylating agents was identified by multivariate linear regression analysis as an independent predictor of a higher density of atretic follicles, whereas increasing age of the patient predicted a better outcome with less follicle atresia and a higher density of maturing follicles. Conclusion This study provides quantitative in vitro evidence of the impact of chemotherapy on developmental capacity of cryopreserved human ovarian tissue. The results indicate that fertility preservation should be carried out, if possible, before initiation of alkylating agents in order to guarantee better in vitro survival of ovarian follicles. In addition, ovarian samples from younger girls show lower viability and fewer developing follicles in culture.

Minimal residual disease of leukemia and the quality of cryopreserved human ovarian tissue in vitro

Auto-transplant of cryopreserved ovarian tissue in leukemia patients carries a risk to reintroduce malignant cells. Maturation of ovarian follicles in vitro is a promising strategy to overcome the leukemic cell contamination. The follicle development and survival in 14 cryopreserved ovarian tissues with leukemia-specific PCR marker was evaluated after 7 or 14 days culture. Minimal residual disease (MRD) quantification was assessed by real-time quantitative PCR in order to identify the MRD positive (n = 6) and negative (n = 8) samples and to monitor levels of MRD before and after culture. The morphology of ovarian follicles were studied by light microscopy. After culture, no statistical significant differences were detected in follicle densities between MRD positive- and negative samples. Ovarian MRD either decreased below undetectable or fluctuated near the baseline level after 7 and 14 days in culture. This study provides quantitative in vitro evidence that leukemia contamination does not affect the follicle survival in cryopreserved ovarian tissue.

Transcription Activation Of Early Human Development Suggests DUX4 As An Embryonic Regulator

In order to better understand human preimplantation development we applied massively parallel RNA sequencing on 337 single cells from oocytes up to 8-cell embryo blastomeres. Surprisingly, already before zygote pronuclear fusion we observed drastic changes in the transcriptome compared to the unfertilized egg: 1,804 gene transcripts and 32 repeat elements become more abundant, among these the double-homeobox gene DUX4. Several genes previously identified as DUX4 targets, such as CCNA1, KHDC1L and ZSCAN4, as well as several members of the RFPLs, TRIMS and PRAMEFs1 were accumulated in 4-cell stage blastomeres, suggesting DUX4 as an early regulator. In the 8-cell stage, we observed two distinct cell types – a transcript-poor cell type, and a transcript-rich cell type with many Alu repeats and accumulated markers for pluripotency and stemness, telomere elongation and growth. In summary, this unprecedented detailed view of the first three days of human embryonic development reveals more complex changes in the transcriptome than what was previously known.