Dr. Hans Snoeck Research
The research program of the Snoeck lab primarily focuses on hematopoiesis with the aim to improve bone marrow transplantation and gene therapy targeted at hematopoietic stem cells (HSCs), and gain insight in leukemogenesis. To achieve a deeper understanding of the mechanisms involved in self-renewal of HSCs, genes underlying quantitative genetic variation in the behavior of HSCs among inbred mouse strains were mapped. After identification of allelic variation in the Prdm16 gene as one of the underlying mechanisms, the lab now focuses on the mechanism of action of Prdm16 in the renewal of HSCs. Expansion of this program into directed differentiation of human embryonic stem cells (ESCs) and induced pluripotent state cells (iPSCs) (collectively called human pluritpotent cells (hPSCs)) arose from the desire to attempt to alleviate post-bone marrow transplantation immune deficiency, caused, among others, by defective T cell reconstitution. Furthermore, the capacity to generate thymic epithelial cells from human pluripotent stem cells will allow the generation of the next generation of ‘personal immune’ mice. As the thymus develops from the anterior foregut endoderm, the approaches we developed also led to strategies to generate virtually every type of cell of the respiratory system from hPSCs.
Hematopoietic stem cells
HSCs reside in the bone marrow (BM), are relatively quiescent, and can self renew and generate all lineages of the hematopoietic system. Despite significant progress in our understanding of mechanisms involved in self-renewal, differentiation and quiescence of HSCs, a coherent picture of how these mechanisms act in concert to regulate steady-state function and homeostatic responses of HSCs in vivo has not emerged yet.Self renewal of HSCs in vitro has not been achieved, while there is overwhelming evidence that HSC self-renewal occurs in vivo. This implies that despite the identification of dozens of cytokines that can affect hematopoiesis, novel insights and more innovative approaches are required. It is therefore critical to understand the mechanism of action of genes that appear specifically required for HSC maintenance and renewal in vivo. To identify such genes, we used quantitative genetics.
Among inbred mouse strains there is extensive genetically determined variation in the function and kinetics of hematopoietic stem and progenitor cells. These traits vary continuously across genetically different individuals as they are determined by the contribution of multiple loci, called quantitative trait loci (QTL). The identification of genes underlying QTL is a potentially powerful approach to reveal novel regulatory mechanisms. Furthermore, as many genes or pathways that show quantitative genetic variation in the mouse model also do so in humans, this approach may allow insight into human genetic variation in a more targeted fashion than is currently possible in genome-wide association studies. We found, through quantitative trait mapping, that allelic variation in the Prdm16 gene plays a role in genetic variation in the hematopoietic system of inbred mouse strains. PRDM16 is a 140kDa zinc finger protein that was originally discovered as a fusion partner in relatively rare translocations in acute myeloblastic leukemia (AML). Elevated PRDM16 expression is frequently observed in karyotypically normal AML, often accompanied by mutations in nucleophosmin. Prdm16 is a frequent target of insertional mutagenesis in mice, causing deletion of the PR-domain.Prdm16 is also expressed in thermogenic brown adipose tissue, and is essential for its development. We showed that allelic variation in Prdm16, consisting of in a single amino acid substitution, affected the response of progenitor cells to the hematopoietic cytokine, Flt3 ligand. However, deletion of Prdm16 causes a profound defect in HSC self-renewal and maintenance. These effects cannot be explained by role of Prdm16 in regulating Flt3 responsiveness of progenitors, because HSCs do not express Flt3 and deletion of Flt3 does not affect HSC function in the mouse.
Current efforts in the laboratory are aimed at identifying the mechanisms of action of Prdm16 in regulating HSC self-renewal. Since Prdm16 is one of a small group of transcription factors that specifically affect HSC renewal, elucidating its mechanism of action will provide a deeper understanding of this still elusive process. Furthermore, we are investigating the role of Prdm16 allelic variation, which is also present in humans, in leukemia, as we have evidence that Flt3 signaling may play a pathogenetic role in at least some forms of acute leukemia.
Generation of thymic epithelial cells from human pluripotent stem cells
The thymus is the site of production of T cells, essential components of the adaptive immune system, from hematopoietic precursors that seed this organ from the bone marrow. The thymus consists of a hematopoietic, an epithelial and a mesenchymal compartment. The hematopoietic component consists of developing T cells and dendritic cells. The epithelial component includes thymic epithelial cells (TECs) of two predominant, but still heterogenous subtypes, cortical (cTEC) and medullary (mTEC), which form a complex three-dimensional structure together with endothelial cells, neural elements, neural crest-derived pericytes, and adipocytes.
A major reason to generate TECs from hPSCs is the improvement of so-called personal immune system (PI) mice, where the human immune system is modeled in the mouse, a major challenge in immunology.Using iPSCs, PI mice can be constructed where thymus and hematopoiesis are autologous and patient-specific, thus capturing genetic diversity in disease susceptibility and immune responses. Furthermore, co-transplantation of autologous, iPS-derived tissues, for example pancreatic beta cells from type I diabetes patients, will allow study of tissue-specific immune responses. Inaddition, there are several medical conditions where thymic replacement would be beneficial. Congenital diseases where a thymus is lacking, such as the FOXN1-deficient nude/Scid syndrome and the DiGeorge syndrome (DGS) are good candidates for thymus replacement therapy. Additional applications of hiPSC-derived thymic tissue are hematopoietic stem cell transplantation (HSCT), age-associated thymic atrophy and autoimmune disease.
The thymus is derived from the posteroventral aspect of the 3rd pharyngeal pouch, and is therefore of endodermal origin. Following developmental paradigms, directed differentiation of TECs should proceed by generation of definitive endoderm, followed by patterning into anterior foregut endoderm. Subsequently, ventrolateral pharyngeal endoderm, 3rd pharyngeal pouch and then TEC progenitor fates should be specified. We have shown that dual inhibition of TGF-b/BMP signaling of hPSC-derived definitive endoderm led to the quantitative generation of anterior foregut endoderm, that could be subsequently differentiated into cells expressing markers of thymus, parathyroids and lung. We further optimized this strategy by devising a strategy to more finely specify pharyngeal endoderm and to induce high expression of the TEC markers FOXN1, b5T and AIRE.
Development of respiratory epithelium from human pluripotent cells
Lung disease kills 120,000 people in the US every year. For many end-stage lung diseases, transplantation is a valid therapeutic option, that is, however, hampered by low availability of donor organs, and surgical, medical and immunological complications. Although adult lung stem and progenitor cells have been identified in the mouse, engraftment of such cells in vivo in injured lungs has not been demonstrated. Likely the best future way to treat severe lung disease is transplantation with engineered lungs consisting of a decellularized lung matrix seeded with patient-specific, autologous lung and airway cells. While extracellular matrix could be derived from donor lungs, the capacity to regenerate lung tissue from autologous cells would therefore constitute a major medical advance.
One way to accomplish this is differentiation of human iPSCs into respiratory epithelial cells and/or into putative postnatal stem cells of the respiratory system. In addition, the ability to generate a diverse array of cell types of the respiratory system will allow disease modeling in vitro, drug testing for treatment of developmental lung abnormalities, as well as testing teratogenic effects of drugs on human lung development. The lung develops from the ventral aspect of the anterior foregut endoderm. At E9.5 in the mouse a tracheal anlage and two lung buds separate from the ventral aspect of endodermal primitive gut tube. Through a complex process of branching morphogenesis, airways develop. This is followed by alveolar morphogenesis, which proceeds in part postnatally and goes through defined pseudoglandular, saccular and vesicular stages. During early postnatal development, lung and tracheobronchial stem cells that provide extensive regenerative capacity are laid down as well.
During our studies into the generation of anterior foregut endoderm from hPSCs, we developed a strategy to specify cells committed to a lung and airway fate with almost 100% purity without the use of reporter genes, and to differentiate these into virtually every cell type of the respiratory system (ciliated cells, mucus cells, secretory (Clara) cells, basal cells, alveolar epithelial type I and type II cells). Further efforts are aimed at determining how specific lineages can be enriched and how postnatal lung stem cells can be generated from hPSCs.
Yvan-Charvet L, Pagler T, Gautier EL, Avagyan S, Siry RL, Han S, Welch CL, Wang N, Randolph GJ, Snoeck HW, Tall AR. (2010). ATP binding cassette transporters and HDL suppress hematopoietic stem cell proliferation. Science, 328:1989-1993.
Avagyan S, Amrani Y, Snoeck HW. (2010) Isolation and analysis of mouse hematopoietic stem cells. Methods Enzymol., 476:429-447.
Aguilo F, Avagyan S, Labar A, Sevilla A, Lee DF, Kumar P, Lemischka IR, Zhou BY, Snoeck HW. (2011). Prdm16 is a physiological regulator of hematopoietic stem cells. Blood, in press.
Green M, Chen A, Nostro MC, d’Souza S, Schaniel C, Lemischka IR, Gouon-Evans V, Keller G, Snoeck HW. (2011). Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nat. Biotechnol., 29:267-272.
Green M, Snoeck HW. (2011) Novel approaches for immune reconstitution and adaptive immune modeling with human pluripotent stem cells. BMC Medicine 9:51.
Avagyan S, Aguilo F, Kamezaki K, Snoeck HW (2011). Quantitative trait mapping reveals a regulatory axis involving peroxisome proliferator-activated receptors, PRDM16, transforming growth factor-b2 and FLT3 in hematopoiesis. Blood, 118:6078-6086.
Longmire TA, Ikonomou L, Hawkins F, Christodoulou C, Cao Y, Jean JC, Kwok LW, Mou H, Rajagopal J, Shen SS, Dowton AA, Serra M, Weiss DJ, Green MD, Snoeck HW, Ramirez MI, Kotton DN. (2012) Efficient derivation of purified lung and thyroid progenitorsfrom embryonic stem cells. Cell Stem Cell, in press.