NORDFERTIL Research Lab Stockholm - projects
Strategies to mature pluripotent stem and immature germ cells into mature gametes
Late side effects affecting future fertility in children undergoing gonadotoxic treatments do still exist. So far, no treatment can be offered to rescue fertility in those patients. In the past, major efforts have been put into optimizing in vivo and in vitro methods for maturation of immature germ cells, but these techniques still remain experimental. For the in vivo methods the immature testicular tissue or spermatogonial stem cells (SSCs) could be transplanted back to the patient after cancer treatment, however in such case there is a potential risk of introducing cancer cells back to the patient. Transplanting the tissue or the isolated SSCs to other animal species for maturation is also an option but then there is a potential risk of introducing the germ cells to xenogenic tissue hosting unknown viruses such as retroviruses. Therefore, such methodology would presently not be considered acceptable for clinical practice.
For in vitro methods, the SSCs could be differentiated in a monolayer on supporting feeder layer or on extracellular matrixes. Also it has been reported that pre-meiotic germ cells from mice can be differentiated in soft agar culture system (SACS) where the cells have 3D support. In addition to those 3D culture approaches, in 2011, Japanese researchers demonstrated for the first time viable offspring from in vitro generated murine sperm using an organ culture. This experiment demonstrates a big step towards the establishment of clinical tools to use in vitro differentiated gametes as potential fertility preservation for young cancer patients. More studies are required to meet efficiency/safety concerns and translate results to the situation in humans.
Jan-Bernd Stukenborg, Assoc Prof, PhD, Group leader
Hormonal effects in the female reproductive tract
The focus for my part of our projects is on hormonal effects and cross-talk between sex steroid hormone receptors and growth factors. The classical steroid hormone receptors are predominantly located in the nucleus, but in recent years there have been evidence also of membrane bound steroid hormone receptors e.g. GPER (G-protein bound estrogen receptor 1), PGRMC (progesterone receptor membrane component) 1 and PGRMC2. These membrane bound receptors could explain some of the very fast effects seen after hormone treatment, which cannot be mediated via the classical nuclear receptors since nuclear/genomic signaling requires longer time.
Lena Sahlin, Assoc Prof, PhD
The origin and fate of human germ cells
Our group is working on the field of fertility preservation and aims to explore the mechanism of development, specification and differentiation of male testicular gonadal cells and understanding the testicular niche.
The overall aim of my project is to establish robust in vitro conditions to investigate cellular and molecular mechanism differentiating pluripotent human stem cells towards the male germ cell lineage. Exploring the potential of various undifferentiated hES cells to differentiate towards specific cell types important for human gonadal tissue and optimizing the culture conditions useful for differentiation of hES cells into male germ cells and testicular somatic cells in vitro are the main focus of the study.
This project will generate a large body of basic information on early human gametogenesis and its regulation which has been extremely difficult to study earlier. It will offer new research models of human gametogenesis and its failures. It will provide new information to understand the cellular and molecular mechanisms of fertility and thereby give insights how to treat male infertility.
- Characterization of human embryonal stem (hES) cells
- Differentiation of hES cells to primordial germ cells
- Differentiation of hES cells to testicular somatic cells
- Study of the original germ layer of primordial germ cells
Halima Albalushi, MD, PhD student
Human induced pluripotent stem cells differentiation towards the male germ cell lineage
Despite the research progress in the andrological area, detailed knowledge about the early development and the biology of male fertility is still inadequate. Studying the human germline development and maturation using fetal, prepubertal and adult testicular tissue as well as pluripotent stem cells can contribute to a better understanding of in vivo as well as in vitro gametogenesis processes.
Further we believe that this knowledge will help to understand infertility causing disorders and the poor outcomes in assisted reproduction.
Therefore my project focuses on one side in understanding the establishment and role of the tubular basement membrane and its function in germ cell specification, maintenance and differentiation.
On the other side I also focus on the X chromosome inactivation status of Klinefelter syndrome (47, XXY) derived iPSCs and the role of the additional X chromosome in germ cell specification, maintenance and differentiation.
- Derivation of clinical grade hiPSC lines from healthy (46,XY) and Klinefelter syndrome (47,XXY) dermal fibroblasts
- Effect of laminin 521 on the pluripotency behavior of hESC
- Investigation of aspects the testicular stem cell niche
- Influence of differentiation techniques for differentiation of hiPSCs towards male germ cells
Magdalena Kurek, MSc, PhD student
Qualitative assessment of fresh and cryopreserved ovarian tissues from pediatric oncological patients
Cryopreservation of ovarian tissue is the only option to preserve fertility in childhood cancer patients facing highly toxic treatments. Chemo- and radiotherapy can damage the ovary, causing permanent infertility in 16% to 41% of girls.
Cryopreserved ovarian tissues can be transplanted back to the patient and are able to restore ovarian function. However, this option is not safe for certain malignancies at high risk of ovarian contamination (e.g. leukemia, Ewing sarcoma).
In vitro follicle growth and oocyte maturation has emerged as a safe alternative method but is still burdened by low efficacy rates, especially for younger patients. Age-related factors, cryostatic cell damage as well as chemotherapeutic agents to which patients have been exposed, have an impact on the oocyte viability in vitro. To date knowledge in this field remains limited, due to the difficulty of obtaining tissues for research from this population.
My research project includes 2 sets of tissues harvested from girls affected by cancer (age range 1-17 yrs):
1) Freshly formalin-fixed ovarian tissues collected after the initiation of chemotherapy (n=35). Expression of key factors involved in follicle activation, maturation and survival will be determined and related to individual patient's characteristics and chemotherapeutic regimens (methods: light microscopy).
2) Frozen ovarian tissues that have been cryopreserved for fertility preservation purposes after the initiation of chemotherapy (n=17), The effects of the cryopreservation process and the effect of chemotherapy on pathways regulating oocyte viability will be assessed in an in vitro culture system (methods: light microscopy, transmission electron microscopy, qPCR, Western Blot).
This project will provide new understandings on the impact of cancer treatments and tissue processing on the in vitro developmental potential of ovarian tissues harvested for fertility preservation purpose in pediatric cancer girls.
Valentina Pampanini, MD, PhD student
Novel culture conditions for human in vitro spermatogenesis
My focus is the generation of experimental models that permits the investigation of the mechanisms of spermatogonial stem cell (SSC) differentiation and self-renewal in vitro. We have recently created a 3D system that allows the generation of testicular organoids and with this the opportunity to explore the role of essential cells and factors regulating the SSC niche and testicular development. The future translation of our knowledge about SSC propagation and differentiation in vitro to the human context will represent new approaches for the treatment of male infertility.
Key techniques: 3D culture, organoid culture, whole-mount straining, confocal microscopy.
Key words: Testicular organoids, SSC niche, testicular development
- Exploring the effect of various three-dimensional (3D) techniques on early gonadal somatic cell maturation in vitro.
- Exploring the effect of previous established three-dimensional gonadal somatic cell culture techniques on early germ cell maintenance in vitro.
- Exploring the effect of signalling agents, such as BMPs, SCF, IGF, FGF and hCG, on germ cell differentiation into meiotic and post-meiotic developmental stages using the previously created human gonadal microenvironment in vitro.
- Exploring the genetic profile of in vitro differentiated germ cells from fresh and cryopreserved testicular tissue.
João Pedro Alves-Lopes, MSc, PhD student
Group leader NORDFERTIL Research Lab Stockholm: