Stem Cell Therapies for Reproductive and Genetic Disorders

Research Group Leader(s)

Research Group Leader: Dr David Cram (Senior Research Fellow)
Deputy Leaders: Dr Gayle Jones, Dr Orly Lacham-Kaplan (Senior Research Fellows)

Overview of the Group’s Research Program

The Group conducts basic research in the fields of infertility and stem cell biology with the aim of translating findings into potential treatments for patients in the clinic. Specific research areas include non-invasive prenatal diagnosis, preimplantation genetic diagnosis, oogenesis, pre- and post implantation development, germ cell development, epigenetics with a focus on embryo health and development to term. The Group has used existing mouse and human embryonic stem cell lines to investigate germ cell development in vitro and for the production of viable sperm and oocytes. More recently, the group has developed and characterized a number of disease-specific human embryonic stem cell lines as new models of inherited genetic disorders such as Huntington Disease and Cystic Fibrosis that will bridge the gap of knowledge gained on the mechanisms of pathogenesis between animal models and patients. In the last five years, the Group has collectively published 36 peer-reviewed papers, including a recent paper in Nature.

Lab Members

Research Assistant

Dr Bi Song, PhD

Higher Degree Students

National and International Collaborators

Professor Steve Bottomley, Dr Melanie Pritchard (Department of Biochemistry and Molecular Biology, Monash University)
Dr. Bayden Wood, Dr Phil Heraud (Centre for Biospectroscopy, School of Chemistry, Monash University)
Dr Adam Hart, Dr James Armitage (Department of Anatomy and Cell Biology, Monash University)
Professor Jock Findlay, Professor Rob McLachlan (Prince Henry's Institute of Medical Research)
Dr Mirella Dottori (Centre for Neurosciences, Department Pharmacology, The University of Melbourne)
Professor Alan Trounson (Director of the California Institute of Regenerative Medicine, US)
Dr Luca Gianaroli (SISMER IVF, Italy)
Dr Carlos Simon  (Fundación Instituto Valenciano de Infertilidad, University of Valencia Spain)
Dr Kostos Pantos (Athens IVF, Greece)

Funding

The Group receives funding for research from a variety of sources, including Monash IVF, the NHMRC and the NIH as well as philanthropic organizations.

The Circles of Life

(Click image above to enlarge -1024 x 819)

Dr David Cram
Senior Research Fellow
Ph: 61 3 99050778
david.cram@med.monash.edu.au

Dr Cram has a major research interests in the development of new technologies for the diagnosis of genetic disease, the identification of new genetic causes of human infertility and the characterization disease-specific and patient-specific stem cells. Much of the outcomes of his research has already been translated into clinical practice with the establishment of a national Y chromosome testing service and preimplantation genetic diagnosis at Monash IVF. He is also actively involved in educating genetic support groups and the community on assisted reproductive technologies, the controversial practice of pre-implantation genetic diagnosis and stem cell technologies for treatment of debilitating diseases. In 2007, he was invited by the journal Nature to validate the first report of primate stem cells derived by somatic nuclear transfer, leading to a publication in Nature. More recently, he has been instrumental in the characterization of new stem cell models of genetic disease to better understand mechanisms that initiate pathology and for drug screening with the ultimate aim of developing effective therapeutics for patients.

Neuronal differentiation. HD negative HES3 (A-D) and HD positive SI-186 (E-H) plated neurospheres immunostained to detect primary neurons using ?-III-tubulin (green) and MAP2ab (red) antibodies, and nuclei stained using Dapi (blue). C and G overlays show co-localization of MAP2ab and ?-III-tubulin staining within primary neurons. I-L, negative controls.

Dr Gayle Jones
Senior Research Fellow
Ph: 61 3 99050779
gayle.jones@med.monash.edu.au

Dr Jones has major research interests in follicularogenesis, oogenesis, preimplantation development, implantation and embryonic stem cells with a focus on the human. Her research has led to improvements in assisted reproduction technologies at Monash IVF such as the development of oocyte maturation and embryo culture media, blastocyst culture and blastocyst biopsy for genetic diagnosis.  More recently, she has developed microarray based techniques and bioinformatic analyses that now allow the reliable and accurate analysis of gene expression to identify molecular changes during oogenesis and pre-implantation development. Using these techniques, she has identified key changes in the transcriptome that discriminate between developmentally competent and non-competent oocytes and pre-implantation embryos. Her current research focus is to understand the basic biology of the human oocyte and the effect of 'disease states' such as diabetes, obesity, age on oocyte quality and to develop strategies to manipulate surplus human oocytes from an IVF cycle to improve the efficiency of reprogramming and somatic cell nuclear transfer.

Blastocyst trophectoderm (TE) biopsy. Micrographs of a sibling cohort of blastocysts pre- and post-TE biopsy that resulted in two live-births following blastocyst transfer. (A-C) Sibling blastocysts on Day 5 post-zona opening and immediately prior to TE biopsy. (D-F) Sibling blastocysts on Day 6 post-biopsy and culture overnight and immediately prior to transfer.

Dr Orly Lacham Kaplan
Senior research Fellow
Ph: 61 3 99050781
orly.lacham-kaplan@med.monash.edu.au

Dr Lacham Kaplan has major research interests in the use of mouse and human stem cells for reproductive therapies. Her research has focused on the production of germ cells, sperm and oocytes, from stem cells. As a reproductive biologist and clinical embryologist, she has been involved in developing cutting edge technologies to overcome fertility problems associated with incompetent gametes. Hence, she has been instrumental in development and implementation of micromanipulation procedures which are now used in IVF critiques. In addition she has been involved in development and optimization of somatic nuclear transfer techniques leading to the production of the first cloned calf in Australia. In recent years she has been focusing on producing artificial gametes in vitro. This includes the haploidization technique where somatic cells are used to fertilize oocytes. More recently, she derived ovarian structures containing oocytes from human embryonic stem cell lines. Her laboratory is one of four in the world leading research in this area and is internationally recognised as an authority in the field of germ stem cells. Currently, she is researching the possibility of using in vitro matured and aged oocytes as an alternative to metaphase II oocytes to produce stem cell lines and has already applied proteomics and Synchrotron based spectroscopic methods to map the biochemical difference between competent and non-competent oocytes.

A. Ovarian structures developed from stem cell derived embryoid bodies.	B. Follicles in the ovarian structures contain "putative oocytes"

Current Research Projects

• Identification of new causes of male and female infertility
• Improving assisted reproduction treatments through an understanding of the biochemical and molecular properties of developmentally competent human egg and embryo
• Understanding the effect of  'disease' states such as obesity and diabetes on egg quality and reproductive fitness
• Derivation and functional analysis of sperm and eggs derived from stem cells
• Derivation and characterisation of disease-specific stem cell lines as useful in vitro models of neurodegenerative disorders
• Application of stem cell lines for treatment of reproductive and genetic disorders 
  
  

Group Publications, 2006-2008

Lacham-Kaplan, O. and A. Trounson "Reduced developmental competence of immature, in-vitro matured and postovulatory aged mouse oocytes following IVF and ICSI" in Reproductive Biology and Endocrinology (2008), 6:58 doi:10.1186/1477-7827-6-58. This work was supported by the NIH.

Marques-Mari AI, Lacham-Kaplan O, Pellicer A, Simón C  (2008) Stem cells in reproductive medicine. Accepted for Human Reproduction Update.


Lacham-Kaplan O, Nayernia K (2008) in-vitro derived germ cells: Therapeutic potentials and risks. Accepted for Human Reproduction Update.

McLachlan, R.I., R.J. Aitken, D. Cram, C. Krausz, and M. O'Bryan, LETTER TO THE EDITOR: Need for Standardization and Confirmation of STS Deletions on the Y Chromosome. Fertil Steril, 2008. 90: 463-464

Niclis J, Trounson AO, Dottori M, Ellisdon A, Bottomley S, Verlinsky Y, Cram DS (2008)  Disease-specific embryonic stem cells: a model for Huntington's Disease. Accepted for Reproductive Biomedicine Online.

D Saiti and O Lacham-Kaplan (2008): Density Gradients for the Isolation of Germ Cells from Embryoid Bodies. Reprod Biomed Online, 2008. 16: 730-40.

*Jones GM, *Cram DS, Song B, Kokkali G, Pantos K, Trounson AO.  (2008) Novel strategy for identifying developmentally competent IVF blastocysts.  Human Reproduction 23: 1748-1759. 
* Equal first authors


Jones GM, Cram DS, Song B, Magli MC, Gianaroli L, Lacham-Kaplan O, Findlay JK, Jenkin G, Trounson AO (2008) Gene expression profiling of human oocytes following in vivo or in vitro maturation.  Human Reproduction 23: 1138-1144.


Saiti D, Lacham-Kaplan O (2007) Mouse germ cells development in-vivo and in-vitro. Biomarker Insight 2:241-252.


West M, Lacham-Kaplan O, Cleary M, Galloway D, Shaw J, Trounson AO, Paris MC (2007). In vitro maturation and intracytoplasmic sperm injection of oocytes collected from hormonally stimulated common wombats, Vombatus ursinus. Animal Reproduction Science 98: 311-321.


Wilson TJ, Lacham-Kaplan O, Gould J, Holloway A, Bertoncello I, Hertzog PJ, Trounson A (2007). Comparison of mice born after intracytoplasmic sperm injection with in vitro fertilization and natural mating. Molecular Reproduction and Development 74: 512-519.


Cram DS, Song B, Trounson AO (2007) Genotyping of Rhesus SCNT pluripotent stem cell lines. Nature 450: E12-14.


Osborne E, Lynch M, McLachlan R, Trounson AO, Cram DS (2007) Microarray detection of Y chromosome deletions.  Reproductive Biomedicine Online 15: 673-680.


Salvado C and Cram DS (2007). Microarray technology for mutation analysis in low template DNA samples. Methods in Molecular Medicine 132:153-174.


Cram DS, Pope A (2007) Preimplantation genetic diagnosis: current and future perspectives. Journal of Law and Medicine 15: 36-44.


Munne S, Gianaroli L, Tur-Kaspa I, Magli C, Sandalinas M, Grifo J, Cram DS, Kahraman S, Verlinsky Y, Simpson JL (2007) Sub-standard application of preimplantation genetic screening may interfere with its clinical success. Fertility and Sterility 88: 781-784.


Kokkali G, Traeger-Synodinos J, Vrettou C, Stavrou D, Jones G, Cram DS, Evangelos M, Trounson AO, Kanavakis E, Pantos K (2007) Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation diagnosis of B thalassemia: a pilot study. Human Reproduction 22: 1443-1449.


Jones GM, Song B, Cram DS, Trounson AO (2007) Optimization of a microarray based approach for deriving representative gene expression profiles from human oocytes. Molecular  Reproduction and Development 74: 8-17.


Loren J, Lacham-Kaplan O (2006)  The employment of strontium to activate mouse oocytes: effects on spermatid-injection outcome. Reproduction131: 259-267.


Lacham-Kaplan O, Chy H, Trounson A (2006) Testicular cell conditioned medium supports differentiation of embryonic stem cells into ovarian structures containing oocytes. Stem Cells 24: 266-273.


Thouas GA, Trounson AO, Jones GM  (2006) Developmental effects of sublethal mitochondrial injury in mouse oocytes.  Biology of Reproduction 74:969-77.


Cram DS, Osborne E, McLachlan RI (2006) Y chromosome microdeletions - implications for assisted conception. Medicine Journal of Australia 185: 433-434.


Davis T, Song B, Cram DS (2006) Successful preimplantation diagnosis of familial adenatomatous polyposis. Reproductive Biomedicine Online 13: 707-711.

Group Publications, 2003-2005

Thouas, GA, Trounson, AO, Jones, GM  (2005)  Effect of female age on mouse oocyte developmental competence following mitochondrial injury.  Biology of Reproduction 73:366-373


Mantzaris D, Cram DS, Howlett D, Kovacs G (2005) Preliminary report: Correct diagnosis of sex in fetal cells isolated from cervical mucus during early pregnancy. Royal Australian and New Zealand Journal of Obstetrics and Gynaecology 45: 529-532.


Lynch M, Cram DS, Reilly A, O'Bryan M, Baker HWG, de Kretser DM, McLachlan R (2005) The Y chromosome gr/gr subdeletion is associated with male infertility.  Molecular Human Reproduction 11:507-512.


Kokkali G, Vrettou C, Traeger-Synodinos J, Jones, GM, Cram DS, Stavrou D, Trounson AO, Kanavakis E, Pantos, K (2005) Pregnancy achieved following trophectoderm biopsy for preimplantation diagnosis of B-thalassemia. Human Reproduction 20: 1855-1859.


Osborne E, Trounson A, Cram DS (2005). A QF-PCR system to detect chromosome 13 aneuploidy from as few as ten cells. American Journal of Medical Genetics 134: 33-38.


Katz-Jaffe MG, Trounson A, Cram DS (2005) Chromosome 21 mosaic human preimplantation embryos predominantly arise from diploid conceptions. Fertility and Sterility 84: 634-643.


Katz GM, Mantzaris D and Cram DS (2005) DNA identification of fetal cells in cervical mucus: potential for non-invasive prenatal diagnosis. British Journal of Obstetrics and Gynaecology 112: 595-600.


Lacham-Kaplan O  (2004): In-vivo and in-vitro differentiation of male germ cells in the mouse. Reproduction 128:147-152.


Thouas GA, Trounson AO, Wolvetang EJ, Jones GM  (2004)  Mitochondrial dysfunction in mouse oocytes results in preimplantation embryo arrest in vitro.  Biology of Reproduction 71:1936-1942.


Cram DS, Lynch M, O'Bryan M, Salvado C, McLachlan R, deKretser DM (2004) Genetic screening of infertile men. Reproduction, Fertility and Development 16: 573-580.


Katz-Jaffe MG, Trounson AO, Cram DS  (2004) Mitotic errors in chromosome 21 of human preimplantation embryos are associated with non-viability. Molecular Human Reproduction 10:143-147.


Lacham-Kaplan O, Shaw J, Sanchez Partida LG, Trounson A  (2003) Oocyte activation after intracytoplasmic injection with snap-frozen sperm results in live offspring from inbred and hybrid mouse strains. Biology of Reproduction 69: 1683-1689.


Tecirlioglu RT, Lacham-Kaplan O,Trounson O (2003)  Live offspring from electro ejaculated mouse sperm. Reproduction, Fertility and Development 14: 471-478.


Thouas GA, Jones GM, Trounson AO  (2003) The 'GO' system  - A novel method of micro-culture for the in vitro development of mouse zygotes to the blastocyst stage.  Reproduction 126:161-169.


Shaw J, Jones GM  (2003) Terminology associated with vitrification and other   cryopreservation procedures for oocytes and embryos. Human Reproduction Update 9:583-605.


Huynh K, Jones G, Thouas G, Britt KL, Simpson ER, Jones MEE. (2003)  Estrogen is not directly required for oocyte developmental competence. Biology of Reproduction 70:1263-1269.


Cram DS, Song B, Trounson AO (2003) Preimplantation diagnosis of Lesch-Nyhan using mini-sequencing primer extension.  Reproductive Biomedicine Online 7:342-345.