The pluripotent potential of embryonic stem cells (ESCs) is frequently exploited to study organogenesis in vitro. The lab has previously carried out critical research in the mouse model concentrating on expansion and differentiation of ESCs and identification of essential regulatory pathways. I have established protocols for the in vitro differentiation of murine ESCs into mineralized osteoblasts (Fig. 1) and matrix-secreting chondrocytes. Not only did these analyses reveal the fact that functioning skeletal cell types had been generated from ESCs, but also resulted in a comprehensive analysis of the expression of bone marker genes and osteo-specific enzymatic activity. From these analyses I hypothesize that osteogenesis in ESCs traverses through four distinct phases: proliferation (I), early specification (II), matrix deposition (III) and mineralization (IV).
A major goal of this research group is a) to embark on deepening the knowledge of stem cell specification into osteogenic lineages, b) to understand molecular regulators of normal bone development and thereby c) to deduce novel therapeutic targets, which could be aimed at during disease intervention in the clinic.
Soluble factors influence the Wnt/CatnB pathway during differentiation. Nitric oxide (NO) agonists or antagonists may both support osteogenesis in ESCs depending on the timing of their administration. NO donor administration aids mineralization and maturation of the ESCs in late differentiation (phase IV), whereas inhibition of the NO pathway supports specification into an osteoprogenitor state (phase III). Further work has shown that a critical phase exists during osteogenesis, in which the addition of NO agonists appears to be necessary for the formation of what would be the equivalent of the primitive streak in vivo [zur Nieden et al., unpublished data], which occurs around day 3 of in vitro differentiation (phase II). Extended analyses have shown that NO influences the localization and expression level of beta-catenin (CatnB), a key player in the Wnt signaling pathway. Timely controlled fluctuations in the CatnB level seem to be essential for osteogenic differentiation and maybe even for the specification of other lineages (Fig. 2).
Elucidating the role of non-canonical Wnts during differentiation. Many of the associated proteins of the non-canonical Wnt pathway (i.e. Wnt5a) have been well characterized in an isolated function as they can interact and signal through other developmental pathways. The key components of the three non-canonical pathways are CamKII, JNK and PKC. As we have established, treatment with Wnt5a during phase III of osteogenic differentiation can support this particular differentiation path. We aim to investigate, which of the three suggested subpathways is involved in the osteogenic process. First data suggests that proliferation of progenitors and the onset of mineralization are modulated by PKCbeta, whereas CamKII seems to modulate cAMP levels and expression of mature bone markers.
Oxidative stress modulates the Wnt/CatnB axis. In contrast to murine ESCs, which can be reasonably well expanded in vitro, primate ESCs tend to differentiate spontaneously in culture. The successful expansion of ESCs therefore might be eased by mimicking the conditions ESCs experience in their natural niche. The lab has begun to explore the role of oxidative stress in ESC maintenance and differentiation. Preliminary results suggest that high glucose levels, which are currently routinely used in ESC culture media, adversely affect stem cell characteristics although supporting proliferation. Interestingly, accumulation of reactive oxygen species (ROS) in osteoblastic cell models can lead to altered cofactor usage by CatnB. I therefore plan to investigate whether ROS are increasingly generated inside the stem cells under high glucose concentrations, which may lead to a divertion of CatnB away from its normally used cofactors, thereby altering cell fate. Future plans include the manipulation of oxygen with subsequent characterization of ROS and apoptosis as well as identification of CatnB cofactors and Wnt pathway members.
MicroRNAs regulate the Wnt/CatnB axis during the osteogenic process. Being signaling molecules, Wnts and NO exert their effects by being translated into intracellular signals ultimately activating the transcriptional machinery. Intriguingly, most of these external factors not only regulate osteogenic processes, but can also be involved in the cellular differentiation into other lineages, such as neurogenesis. Consequently, it is likely that another (master) level of regulation may exist in addition to these external stimuli. This master regulator could be microRNAs (miRNAs). These RNA molecules are short 20–22 nucleotide RNA structures that are negative regulators of gene expression in more than 30% of protein-coding genes in a variety of eukaryotic organisms. Additionally, miRNAs seem to be involved in regulating stem cell organization affecting self-renewal, proliferation and differentiation. In collaboration with the RNomics group of the Fraunhofer Institute for Cell Therapy & Immunology (Germany) the lab has successfully identified 25 miRNAs, which are differentially regulated during the first 8 days of the osteogenic developmental path. Future studies will characterize the role of these miRNAs in more depth and ultimately lead to the identification of molecules, which may be malfunctioning in degenerative and congenital bone disease
HONORS AND AWARDS
2006, 2007 Alumni Award, Canadian Stem Cell Network Centres of Excellence
2007 Nobel Laureate Meeting, Lindau, Germany, Nominated and Selected Participant
2007 IQ Innovation Price Central Germany, Top Ten Finalist
2005 Leica Meritorious Performance Award, University of Calgary
2004 Best Poster Presentation, Canadian Stem Cell Network Annual General Meeting
2003 – 2005 Post-doctoral Fellowship, Alberta Heritage Foundation for Medical Research
See publications on PubMed
Quintana L, zur Nieden NI, Semino CE. Morphogenetic and regulatory mechanisms during developmental chondrogenesis. Tissue Eng Part B, accepted
Davis LA, zur Nieden NI (2008). Mesodermal fate decisions of a stem cell: the Wnt switch. Cell Mol Life Sci 65(17), 2658-2674
zur Nieden NI. Embryonic stem cells for the prediction of developmental toxicity in pharmacological screening. In: Conrad K, Lehman W, Sack U, Schedler U (eds). Multiparameteranalytics - Methods, Applications, Perspectives. Pabst Science Publishers, Lengerich 2008, pp 198-220. Article in German
Meng GL*, zur Nieden NI*, Liu SY, Cormier JT, Kallos MS, Rancourt DE. (2008). Properties of murine embryonic stem cells maintained on human foreskin fibroblasts without LIF. Mol Reprod Dev 75(4), 614-622 (*Authors contributed equally to this study).
zur Nieden NI, Price FD, Davis LA, Everitt R, Rancourt DE (2007). Gene array analysis on mixed ES cell populations: a biphasic role for beta-catenin in osteogenesis. Mol Endocrinol 21(3), 674-685
zur Nieden NI, Cormier JT, Kallos MS, Rancourt DE (2007). Murine embryonic stem cells maintain pluripotency after long-term culture in suspension bioreactors. J Biotechnol 129(3), 421-432
zur Nieden NI, Kempka G, Rancourt DE, Ahr HJ (2005). Induction of chondro-, osteo- and adipogenesis in embryonic stem cells by bone morphogenetic protein-2: Effect of co-factors on differentiating lineages. BMC Developmental Biology 5, 1-15
zur Nieden NI, Kempka G, Ahr HJ (2003). In vitro differentiation of embryonic stem cells into mineralized osteoblasts. Differentiation 71, 18-27