Outi Hovatta's Laboratory
Welcome to our laboratory
Outi Hovatta Professor in Obstetrics and Gynaecology, especially Assisted Conception was appointed professor in 1998. She is active at the Department for clinical science, intervention and technology. Outi was born in 1946 in Finland. She made her undergraduate studies during 1970 and made her dissertation at the Anatomical Institution at the Helsingborg University. Her worked involved testicles myoid cells (cells that coordinates sperm production). She became a specialist in obstetrics and gynecology at the university’s hospital. In 1986 she was appointed at the IVF clinic in Väestöliitto, which is Finland’s biggest infertility clinic, with about 1300 patient treatments per year. In 1983 she got a docent position in andrology and during1995 and 1996 she worked as a senior researcher fellow in London at the Royal Postgraduate Medical School. Between 1997 and 1998 she was a guest research at Imperial College School of Medicine. At the end of 1998 she was appointed Professor in Obstetrics and gynecology at Karolinska Institutet. Her research interest is mainly in infertility problems, the genetics behind it and the generation of optimized treatments for infertility. Maturation of eggs follicles in vitro is one of Outi’s main research areas. Since 2001 she started work in human embryonic stem cells, working specially with the generation of methods that may allow the cells to be use in clinical trials related with cell replacement therapies.
Human oocytes and pluripotent stem cells
Our constellation focuses on demanding cell culture projects aiming at clinical applications. One of them is maturation of human ovarian follicles and oocytes from cryostored human ovarian tissue as fertility preservation methods of women who lose their eggs due to chemotherapy or genetic causes. Since we, for the first time, succeeded in freezing human ovarian tissue (Hovatta et al. Hum Reprod 1996), this method has spread all over the world. The tissue can be transplanted back to the ovary of the same woman when she desires pregnancy (von Wolff et al. Eur J Cancer 2009). 15 children have been born so far. We have improved the cryostorage significantly by applying vitrification of the tissue (Keros et al. Hum Reprod 2009). In situations where there is a possibility of remaining malignant cells in the tissue, re-transplantation is too risky. Therefore, we have been developing a method to culture oocytes all the way from primordial follicles to full maturity. This project has been challenging in human due to the long maturation time. We have detected that TGFβ family growth factors, follitropin, activin and insulin promote follicular growth and survival in vitro. When our collaborators (Reddy et al.Science2003) showed that PI3 kinase signalling pathway in essential in keeping the primordial follicles dormant in mouse ovary, we started studying it in human ovary using our well established tissue cultures. This is the basis of our present efforts to achieve our goal, mature oocytes for in vitro fertilization (IVF) from frozen thawed ovarian tissue. We also want to show that such oocytes and embryos resulting from them are normal.
The second aim has been to derive and culture clean and safe human embryonic stem cells (hESC) starting from supernumerary human IVF embryos. We have since 2002 derived 30 fully characterized hESC lines while at the same time improving the process and the quality of the lines (Ström et al. In Vitro 2010). We were the first to derive hESC using human skin fibroblasts instead of mouse embryonic fibroblasts as feeder cells (Hovatta et al. Hum Reprod 2003), chemically defined culture medium in derivation instead of fetal bovine serum (Inzunza et al. Stem Cells 2005), and mechanical isolation of the inner cell mass instead of immunosurgery involving rodent antibodies (Ström et al. Hum Reprod 2007). A chemically defined clinical grade cryopreservation for hESC and induced pluripotent cells (hiPSC) was developed (Holm et al. Hum Reprod 2010). We were a partner in the EU integrated project ESTOOLS, leading the WP Optimisation and standardization of hESC cultures, resulting in a xeno-free, feeder-free defined culture of hESC and hiPSC (Rodin et al. Nat Biotechnol 2010). We have actively participated international culture media studies (Akopian et al In Vitro 2010), and constructed a new ready-made xeno-free defined medium (Rajala et al. PlosOne 2010) for hESC and hiPSC. From early stages of hiPSC studies, we have been involved in improving also their culture conditions and quality (Unger et al. Hum Repord 2009). Genetic stability studies of hESC and iPSC lines were initiated (Närva et al. Nat Biotechnol 2010, Hovatta et al. PlosOne 2010), and are still central in our program. Our hESC lines have been distributed to more than 100 laboratories, and they are registered in the EU hESC registry.
The third closely related activity has regarded the genetic and epigenetic regulation of human oocytes and early embryonic development in close collaboration with professor Juha Kere’s team. We have access and ethics approval to use donated human oocytes and embryos which cannot be used in patients’ treatment. We have already data based on microarrays (Zhang et al PlosOne 2007, Zhang et al.PlosOne 2008) including follow-up of function of selected genes, and RNA-seq data (ongoing).
Current research activities
Maturation of human ovarian follicles and oocytes in vitro is carried out in the IVF unit of the Karolinska University Hospital Huddinge where we have access to small donated cortical pieces of human ovary. Docent Monalill Lundqvist, PhD student Mona Sheikhi, researcher Jose Inzunza and members of the personnel of the unit are involved in addition to Outi Hovatta who is the project leader. We work in close collaboration with prof Kui Liu and postdoc Deepak Adhikari from Umeå and Evelyn Telfer from Edinburgh. We will further develop the already feasible closed vitrification system, so that we can use only one cryoprotectant, ethylene glycol. The experiments have been initiated and according to the preliminary results, the system will be ready during 2011. Using our established culture system of ovarian cortical slices, and after mechanical isolation of the follicles after four days in order to remove the inhibiting influence of the stromal tissue, we culture the follicles for up to three weeks in a medium which contains a PTEN inhibitor (hopic), activin A, and FSH. After that time, 70% of the follicles reach the antral stage, which is much more often that what was achieved before stimulating the PI3K pathway by removing the inhibition caused by PTEN (Figure).
The 12 day maturation time proved the same as in mouse. We continue these experiments and start isolating the cumulus-oocyte complexes for final maturation of the oocytes in vitro (IVM). The fully grown oocyte is placed to third step culture with follitropin and lutropin. After 36 hours, we aim to fertilize the eggs using intracytoplasmic sperm injection (ICSI). This last step is the one already successfully used in clinical IVM where immature eggs are collected from non-stimulated ovaries (Hreinsson et al. Hum Reprod 2003). We then follow embryonic development until blastocyst stage. Ethics approval has been obtained from the Regional Ethics Board in Stockholm. The obtained embryos are used for analyses of chromosomal and epigenetic normality using high throughput sequencing in the KI core facility in collaboration with prof Juha Kere. If we manage in getting normal embryos we proceed to the first clinical treatment of women who have recovered from leukemia, and who have cryostored ovarian tissue, maybe within three years. When successful, this new method will revolutionize the treatment of infertility due to chemotherapy, but it could also be applied to other groups of women needing IVF.
In collaboration with Kui Liu we are initiating maturation of oocytes from the biopsied donated ovarian cortical tissue. We then activate parthenogenesis, and derive embryonic stem cell lines form the obtained parthenogenic embryos using the same methods as in our earlier hESC derivation. We have ethics approval from the Regional Ethics Board in Stockholm.
Together with prof Christer Höög, KI, we study the causes of aneuploidy in human oocytes during meiotic divisions using donated immature oocytes obtained in stimulated ICSI cycles. Immature oocytes cannot be injected with sperm, and they can be donated for research (ethics approval ok). The meiotic division in vitro is studied by fixing the oocytes at GV,MI and MII stages and studying the chromatids immunohistochemically.
Optimising the clinical treatment of women needing fertility preservation (breast cancer, hereditary disorders) etc is carried out by the specialist in gynaecological endocrinology,MD, PhD Kenny Rodriguez-Wallberg. She and Dr Jose Inzunza who works with genetic stability and characterisation of the hESC, are applying for senior lectureship in 2011.
Dr Kenny Rodriguez-Wallberg is also working in translational research and it aiming at developing novel treatments that may reduce chemotherapy-induced ovarian damage and to further investigate clinical aspects of fertility preservation in female and male cancer patients as well as their emotional needs. The impact of specific conditions in female patients as breast cancer, hereditary breast cancer and ovarian tumours is specifically studied regarding safety of fertility preservation options. The results of her work in the animal experimental studies aim at developing pharmacological ovarian protection treatments. Results of the clinical studies will provide knowledge on safety, risks and effectiveness of fertility preservation methods in female and male patients as well as on psychosocial issues of fertility preservation in both sexes.
Clinical grade hESC lines will be established in the Vecura GMP facility using already established standard operational procedures (SOP), in collaboration with prof Ernest Arenas. Dr Magda Forsberg, and technician Kelly Day will derive GMP-grade human skin fibroblasts from donated skin biopsies in connection of pediatric operations (prof Agneta Nordenskiöld, KI). The fibroblasts are used as feeder cells in derivation of hESC in the defined medium (RegES) which we designed, now produced by Vitrolife for this purpose. These xeno-free GMP grade hESC can then be used in clinical trials, and treatment in the future. The plan is to get the first such lines accepted by the Swedish Medicines Agency in 2012-2013.The first implication will be neurodegenerative disorders. Neural differentiation of dopamine neurons (Ernest Arenas), cholinergic neurons (Agneta Nordberg) and motoneurons (Erik Sundström) using hESC and iPSC is ongoing as collaboration.
The existing 30 hESC lines will be expanded and banked in the new KI core facility for iPSC and ESC. They will be available to researchers who need them. Our laboratory manager, PhD Liselotte Antonsson, who is further optimizing the derivation methods has already started the expansions. Feeder-free culture optimization on new laminins in ongoing with prof Karl Tryggvason’s team with Frida Holm and Kelly Day from our team.
Dr Rosita Bergström has her background in epigenetics, and is comparing histone acetylation and methylation patterns as consequences of different culturing systems. Together with Ph.D students Frida Holm, Rós Kjartansdóttir and Sarita Panula, Rosita Bergström are also working on germ cell differentiation in an international collaboration with Profossor Renee Reijo Pera, Stanford University and Professor Shinya Yamanaka, Kyoto University. Our first manuscript has been accepted for publication in Human Molecular Genetics (Panula et al. Hum Mol Gen 2010). Sarita Panula is now a visiting student in Yamanaka’s laboratory. Germ cell differentiation from hESC and hiPSC offers excellent possibilities for studying human early gametogenesis, particularly the epigenetics involved.
Hepatocyte differentiation from hESC and hiPSC carries a great hope for all patients with liver diseases who are waiting for liver/hepatocyte transplantation. It gives also a good opportunity to understand the early events in human liver development on the molecular level. Dr Rosita Bergström, together with PhD student Mohammed Saliem, performs the work in our laboratory in collaboration with Dr Petter Björqvist, Cellartis AB, Göteborg, Sweden, and Dr Ewa Ellis, KI.
Suvi Asikainen, PhD, is a postdoc who studied microRNA and small interfering RNA-mediated gene regulation in C. elegans in the University of Eastern Finland. She joined us to study the regulation of pluripotency by specific miRNA families in hESCs and hiPSCs. Her aim is to generate hiPSC by reprogramming mesenchymal stem cells by viral-free transfection of commercial microRNA mimics and inhibitors. The studies have been initiated by high-throughput sequencing of microRNAs (Illumina) in our hESC (HS401, HS181) and a partially reprogrammed iPSC (CHiPS A) line to make us able to select rational mimic pools for transfections. The sequencing and bioinformatics analyses are made in collaboration with Iiris Hovatta group, Aalto University, Helsinki, Finland, and Garry Wong group, University of Eastern Finland. iPSCs generated using minicircle DNA vectors will be used in parallel to follow the possible reprogramming of cells. She is also interested of hESC-derived microvesicles and aims to study their suitability to be harnessed as lipid vehicles for miRNA transfections. She has received a three-year postdoc grant from the Academy of Finland.
Future research vision
Establishing differentiation protocols for human pluripotent cells for transplantation using our clinical grade safe cell lines will be reached within the next 5-10 years by our team and the collaborators. The important safety aspects regarding genetic and epigenetic normality, and avoiding tumorigenicity and immune rejection, all in the focus of our research, will be solved in a manner allowing clinical trials in severe diseases. Germ cell development in vitro, starting from stem cells will continue as a key area. Within 5 years of intensive research, we have probably achieved full maturation of oocytes in vitro from primordial follicles, and normal embryonic development to allow clinical trials among recovered cancer patients. The constellation will continue in these and new breaking results, with the next generation senior scientists in the constellation (Bergström, Rodriguez- Wallberg, Antonsson, Inzunza, Forsberg) becoming very capable of developing the projects further with our collaborators.
References. Given in the text
Liselotte Antonsson, PhD.
Liselotte is working as a lab manager in Outi Hovattas stem cell derivation lab. She is involved in developing clinical grade feeder-free, xenofree human stem cell lines, aiming at GMP production and clinical applications. She is also responsible for the bank of human embryonic stem cells, at KI, Huddinge. Liselotte received her PhD in cell and molecular biology in 2007, from the University of Lund, Sweden.
Sonya Piskounova,, MSc
Sonya Piskounova is a postdoctoral fellow in the group of Outi Hovatta since December 2012. She has an MSc in Molecular Biotechnology Engineering (2006) and a PhD in Polymer Chemistry (2011) from Uppsala University. Sonya’s research interests include biomaterials, drug delivery and cell therapies for tissue regeneration. Her current project is on differentiation of human embryonic stem cells into retinal pigmented epithelium cells with the goal to develop a treatment for age-related macular degeneration.