Gender	Male
E-mail	gordon.keller@mssm.edu
Education and Training	Ph.D., University of Alberta
	B.Sc., University of Saskatchewan
	Fellowship, Ontario Cancer Institute

Education and Training	Ph.D., University of Alberta
	B.Sc., University of Saskatchewan
	Fellowship, Ontario Cancer Institute

Development of the hematopoietic, vascular, and cardiac lineages from ES cells

The hematopoietic, vascular, and cardiac lineages develop early in embryonic life from mesoderm populations that are generated in a defined temporal pattern. We have previously identified a unique progenitor called the blast colony-forming cell (BL-CFC) that displays the potential to generate both hematopoietic and vascular progeny. Using the EB/ES model, the BL-CFC arises following two to four days of EB differentiation, expresses the tyrosine kinase receptor vascular endothelial growth factor receptor 2, (VEGFR2 or Flk1) and generates blast cell (hemangioblast) colonies with hematopoietic, endothelial and vascular smooth muscle (vascular potential) in methylcellulose cultures in response to vascular endothelial growth factor (VEGF). These characteristics of the BL-CFC suggest that it represents the equivalent of the hypothetical yolk sac hemangioblast, the common progenitor of the hematopoietic and endothelial lineages. To define the molecular events regulating BL-CFC induction and development, we analyzed targeted ES cells lacking either a functional scl or Runx1 gene. These studies demonstrated that both genes play a pivotal role in early hematopoietic commitment, at the hemangioblast stage of development.

To further investigate the relationship of the BL-CFC to mesoderm, we analyzed the development of these progenitors in EBs generated from ES cells in which the green fluorescent protein cDNA has been targeted to the mesoderm gene, brachyury. Brachyury, the founding member of the T-box family of transcription factors, is expressed in all nascent mesoderm and then down regulated as these cells undergo patterning and specification into derivative tissues. Early EBs generated from the GFP-Bry ES cells were found to contain three distinct populations with respect to GFP (brachyury) and Flk-1 expression: GFP-Flk-1-, GFP+Flk-1- and GFP+Flk-1+. The GFP+Flk-1+ fraction contains the most mature population of cells with the majority of the BL-CFC found within the EB. While the GFP+Flk-1- cells do not generate hemangioblast colonies when plated directly into methylcellulose cultures, they do acquire this potential following an additional 24 hours of liquid culture, suggesting that they represent pre-hemangioblast mesoderm. In addition to BL-CFC, cells within the GFP+Flk-1- fraction also differentiate to cardiomyocytes following additional culture, indicating that this fraction also contains pre-cardiac mesoderm.

Ongoing projects in this area of my research program include: 1) analysis of the regulation of BL-CFC development, 2) characterization of the transition from the hemangioblast stage to the earliest hematopoietic- and vascular-restricted stages of development, 3) characterization of the role of Scl in hematopoietic commitment of the BL-CFC, 4) identification and functional characterization of the hemangioblast in the early yolk sac and the intra-embryonic P-Sp/AGM regions of the mouse embryo, 5) analysis of the role of Notch receptors in the commitment of the BL-CFC to the hematopoietic and vascular lineages, 6) analysis of the role of Epo/EpoR signaling in the establishment of the BL-CFC, 7) identification, isolation and characterization of the hematopoietic stem cell from EBs, and 8) characterization of the pre-hemangioblast and pre-cardiac mesoderm in EBs.

Lineage Specific Differentiation of Embryonic Stem (ES) Cells in Culture

Embryonic stem (ES) cells are totipotent stem cell lines that can be maintained indefinitely in an undifferentiated state in culture by growth on fibroblast feeder cells in the presence of leukemia inhibitory factor (LIF). When removed from these stem cell conditions, ES cells will differentiate and give rise to colonies known as embryoid bodies (EBs) that contain derivatives of the three germ cell layers, ectoderm, mesoderm and endoderm. This capacity of embryonic stem (ES) cells to undergo multilineage differentiation in culture provides an in vitro model system with which to study the developmental biology of germ layer induction and specification. In addition to providing a developmental model, the ES/EB system offers a novel and unlimited source of lineages and tissue for transplantation for future cell replacement therapy. The research program in my lab focuses on understanding the mechanisms that regulate the induction and specification of mesoderm and definitive endoderm using the ES/EB system as a model of embryonic development.

Growth and Differentiation of Human Embryonic Stem Cells

Before the potential of ES cells to form cell-based therapies can be realized, the findings from studies on mouse ES cells must be translated to the human system. To begin these translational studies, we have acquired four different human ES cell lines: HI1, HES2, HES3, and HES4. This aspect of my research program is focused on defining conditions for the efficient induction of mesoderm and endoderm from the human ES cells.

Ongoing Projects

1. Characterization of conditions for the efficient growth and maintenance of human ES cells in culture
2. Differentiation of human ES cells to mesoderm and derivative tissues
3. Differentiation of human ES cells to endoderm and derivative tissues

Keller Lab Web Site

Commitment of ES cells to Endoderm-Derived Lineages

Understanding the mechanisms regulating endoderm induction and tissue specific differentiation has become an area of intense investigation for several reasons. The development of definitive endoderm and its subsequent patterning and differentiation leads to the formation of many major organ systems in the body including liver, pancreas, lung, thyroid, and intestines. In addition, several relatively common and devastating diseases target a number of these derivative tissues. To establish the ES/EB system as a useful model for such studies, it is important to first define conditions that promote efficient endoderm differentiation in these cultures. As an initial approach to investigating the endoderm potential of ES cells, we varied the exposure of the developing EB to fetal calf serum (FCS), the primary source of inducing factors in these cultures. We found that a 48-hour restricted exposure to FCS, followed by four days of culture in serum-free media resulted in the development of a population that expressed markers indicative of definitive endoderm, including HNF3_, Mixl1, Sox17, and Hex. Further culture of these EBs, under conditions known to support the development of hepatocyte-like cells led to the development of cells that expressed genes indicative of hepatocyte development and maturation including _-fetoprotein (AFP), albumin (ALB), transthyretin (TTR), alpha1-antitrypsin (AAT), tyrosine aminotransferase (TAT), and carbamoyl phosphate synthetase I (CPase). To investigate the developmental relationship of the endoderm and mesoderm lineages within the EBs, we analyzed the GFP-Bry+ and GFP-Bry- fractions for endoderm potential. At day three of differentiation, all endoderm potential was found in the GFP-Bry+ fraction. This finding suggests that endoderm and mesoderm may arise from a common mesendoderm progenitor.

Research Project Areas

1. Analysis of the regulation of endoderm induction and specification
2. Identification and characterization of the mesendoderm progenitor
3. Characterization of the development and maturation of the hepatocyte lineage in the ES differentiation cultures
4. Functional analysis of the ES cell-derived hepatocytes

Fehling HJ, Lacaud G, Kubo A, Kennedy M, Keller GM, Robertson S, Kouskoff V. Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 2003 Sep; 130(17): 4217-4227.

Huber TL, Kouskoff V, Fehling HJ, Palis J, Keller G. Haemangioblast commitment is initiated in the primitive streak of the mouse embryo. Nature 2004 Dec; 432(7017): 625-30.

Kubo A, Shinozaki K, Shannon JM, Kouskoff V, Kennedy M, Woo S, Fehling HJ, Keller GM. Development of definitive endoderm from embryonic stem cells in culture. Development 2004 Apr; 131(7): 1651-1662.

Kubo A, Chen V, Kennedy M, Zahradka E, Daley GQ, Keller G. The homeobox gene HEX regulates proliferation and differentiation of hemangioblasts and endothelial cells during ES cell differentiation. Blood 2005 Jun; 105(12): 4590-7.

D'Souza SL, Elefanty AG, Keller G. SCL/Tal-1 is essential for hematopoietic commitment of the hemangioblast but not for its development. Blood 2005 May; 105(10): 3862-70.

Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev 2005 May; 19(10): 1129-55.

Kouskoff V, Lacaud G, Schwantz S, Keller G, Fehling HJ. Sequential development of hematopoietic and cardiac mesoderm during embryonic stem cell differentiation. Proc Natl Acad Sci U S A 2005 Sep; 102(37): 13170-5.