Regulation of Spermatogenesis: Differentiation of GFP-labeled Stem Cells and the Function of Cytoplasmic Bridges

During spermatogenesis, different genes are expressed in a strictly coordinated fashion providing an excellent model to study cell differentiation. Recent identification of testis specific genes and the development of green fluorescence protein (GFP) transgene technology and an in vivo system for studying the differentiation of transplanted male germ cells in infertile testis has opened new possibilities for studying the male germ cell differentiation at molecular level. We have employed these techniques in combination with transillumination based stage recognition (Parvinen and Vanha-Perttula, 1972) and squash preparation techniques (Parvinen and Hecht, 1981) to study the regulation of male germ cell differentiation. By using transgenic mice expressing enhanced-(E)GFP as a marker we have studied the expression and hormonal regulation of beta-actin and acrosin proteins in the developmentally different living male germ cells. Beta-actin was demonstrated in all male germ cells, whereas acrosin was expressed only in late meiotic and in postmeiotic cells. Follicle stimulating hormone stimulated b-actin-EGFP expression at stages I-VI and enhanced the formation of microtubules in spermatids and this way reduced the size of the acrosomic system. When EGFP expressing spermatogonial stem cells were transplanted into infertile mouse testis differentiation and the synchronized development of male germ cells could be observed during six months observation time. Each colony developed independently and maintained typical stage-dependent cell associations. Furthermore, if more than two colonies were fused, each of them was adjusted to one stage and synchronized. By studying living spermatids we were able to demonstrate novel functions for Golgi complex and chromatoid body in material sharing between neighbor spermatids. Immunosytochemical analyses revealed a transport of haploid cell specific proteins in spermatids (TRA54 and Shippo1) and through the intercellular bridges (TRA54). Cytoskeleton inhibitor (nocodazole) demonstrated the importance of microtubules in material sharing between spermatids and in preserving the integrity of the chromatoid body. Golgi complex inhibitor, brefeldin A, revealed the great importance of Golgi complex i) in acrosomic system formation ii) TRA54 translation and in iii) granule trafficking between spermatids.

Pluripotency and genetic stability of human pluripotent stem cells

Pluripotent cells have the potential to differentiate into all somatic cell types. As the adult human body is unable to regenerate various tissues, pluripotent cells provide an attractive source for regenerative medicine. Human embryonic stem cells (hESCs) can be isolated from blastocyst stage embryos and cultured in the laboratory environment. However, their use in regenerative medicine is restricted due to problems with immunosuppression by the host and ethical legislation. Recently, a new source of pluripotent cells was established via the direct reprogramming of somatic cells. These human induced pluripotent stem cells (hiPSCs) enable the production of patient specific cell types. However, numerous challenges, such as efficient reprogramming, optimal culture, directed differentiation, genetic stability and tumor risk need to be solved before the launch of therapeutic applications. The main objective of this thesis was to understand the unique properties of human pluripotent stem cells. The specific aims were to identify novel factors involved in maintaining pluripotency, characterize the effects of low oxygen culture on hESCs, and determine the high resolution changes in hESCs and hiPSCs during culture and reprogramming. As a result, the previously uncharacterized protein L1TD1 was determined to be specific for pluripotent cells and essential for the maintenance of pluripotency. The low oxygen culture supported undifferentiated growth and affected expression of stem cell associated transcripts. High resolution screening of hESCs identified a number of culture induced copy number variations and loss of heterozygosity changes. Further, screening of hiPSCs revealed that reprogramming induces high resolution alterations. The results obtained in this thesis have important implications for stem cell and cancer biology and the therapeutic potential of pluripotent cells.

Regulation of self-renewal and detection of karyotypic changes of pluripotent human embryonic stem cells

Human embryonic stem cells are pluripotent cells capable of renewing themselves and differentiating to specialized cell types. Because of their unique regenerative potential, pluripotent cells offer new opportunities for disease modeling, development of regenerative therapies, and treating diseases. Before pluripotent cells can be used in any therapeutic applications, there are numerous challenges to overcome. For instance, the key regulators of pluripotency need to be clarified. In addition, long term culture of pluripotent cells is associated with the accumulation of karyotypic abnormalities, which is a concern regarding the safe use of the cells for therapeutic purposes. The goal of the work presented in this thesis was to identify new factors involved in the maintenance of pluripotency, and to further characterize molecular mechanisms of selected candidate genes. Furthermore, we aimed to set up a new method for analyzing genomic integrity of pluripotent cells. The experimental design applied in this study involved a wide range of molecular biology, genome-wide, and computational techniques to study the pluripotency of stem cells and the functions of the target genes. In collaboration with instrument and reagent company Perkin Elmer, KaryoliteTM BoBsTM was implemented for detecting karyotypic changes of pluripotent cells. Novel genes were identified that are highly and specifically expressed in hES cells. Of these genes, L1TD1 and POLR3G were chosen for further investigation. The results revealed that both of these factors are vital for the maintenance of pluripotency and self-renewal of the hESCs. KaryoliteTM BoBsTM was validated as a novel method to detect karyotypic abnormalities in pluripotent stem cells. The results presented in this thesis offer significant new information on the regulatory networks associated with pluripotency. The results will facilitate in understanding developmental and cancer biology, as well as creating stem cell based applications. KaryoliteTM BoBsTM provides rapid, high-throughput, and cost-efficient tool for screening of human pluripotent cell cultures.

Autologous Stem Cell Transplantation In Multiple Myeloma

Background. Multiple myeloma (MM) is the second most common hematologic malignancy after lymphomas In Finland: the annual incidence of MM is approximately 200. For three decades the median survival remained at 3 to 4 years from diagnosis until high-dose melphalan treatment supported by autologous stem cell transplantation (ASCT) became the standard of care for newly diagnosed MM since the mid 1990’s and the median survival increased to 5 – 6 years. This study focuses on three important aspects of ASCT, namely 1) stem cell mobilization, 2) single vs. double ASCT as initial treatment, and 3) the role of minimal residual disease (MRD) for longterm outcome. Aim. The aim of this series of studies was to evaluate the outcomes of MM patients and the ASCT procedure at the Turku University Central Hospital, Finland. First, we tried to identify which factors predict unsuccessful mobilization of autologous stem cells. Second, we compared the use of short-acting granulocyte-colony stimulating factor (GCSF) with long-acting G-CSF as mobilization agents. Third, one and two successive ASCTs were compared in 100 patients with MM. Fourth, for patients in complete response (CR) after stem cell transplantation (SCT), patient-specific probes for quantitative allele-specific oligonucleotide polymerase-chain reaction (qASO-PCR) measurements were designed to evaluate MRD and its importance for long-term outcome. Results. The quantity of previous chemotherapy and previous interferon use were significant pre-mobilization factors that predicted mobilization failure, together with some factors related to mobilization therapy itself, such as duration and degree of cytopenias and occurrence of sepsis. Short-acting and long-acting G-CSF combined with chemotherapy were comparable as stem cells mobilizers. The progression free (PFS) and overall survival (OS) tended to be longer after double ASCT than after single ASCT with a median follow-up time of 4 years, but this difference disappeared as the follow-up time increased. qASO-PCR was a good and sensitive divider of the CR patients into two prognostic groups: MRD low/negative (≤ 0.01%) and MRD high (>0.01%) groups with a significant difference in PFS and suggestively also in OS. Conclusions. When the factors prediciting a poor outcome of stem cell mobilization prevail, it is possible to identify those patients who need specific efforts to maximize the mobilization efficacy. Long-acting pegfilgrastim is a practical and effective alternative to short-acting filgrastim for mobilization therapy. There is no need to perform double ASCT on all eligible patients. MRD assessment with qASO-PCR is a sensitive method for evaluation of the depth of the CR response and can be used to predict long-term outcome after ACST.