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KAUFMAN LAB RESEARCH OVERVIEW:
Research in the Kaufman lab in the Stem Cell Institute at the University of Minnesota uses embryonic stem (ES) cells to understand the earliest stages of blood development. Individuals produce billions of blood cells every day. The stimuli and genes that allow individual hematopoietic stem cells (HSCs) to produce mature progeny such as red blood cells, white blood cells, lymphocytes and platelets has been studied in considerable depth and serves as a model system in developmental biology. However, how hematopoietic cell populations are derived during early embryogenesis remains poorly understood, especially in the human system. As outlined, our studies focus on use of human ES cells to investigate various aspects of human blood cell development.
Research in our lab has defined methods to derive multiple types of mature blood cells from human ES cells. As outlined in the figure below in vitro and in vivo models are used to elucidate extracellular protein interactions and intracellular genetic regulation that impact these developmental pathways. Currently, studies are underway to better define the conditions that support hematopoietic cells by addition of specific cytokines and growth factors, overexpression of exogenous genes in the ES cells, and by manipulation of the stromal cells to identify the role of specific proteins.
Another portion of our current research aims to determine the in vivo potential of the human ES cell-derived hematopoietic cells. Proof of an ES cell-derived HSC requires evidence of long-term multilineage engraftment in vivo. Other related aspects involve use of human ES cells to study endothelial cell development and characterization of potential hemangioblast cells that serve as a common precursor to blood and endothelial cells.
Methods of Embryonic Stem Cell Differentiation
Hematopoiesis from Human ES cells
SPECIFIC PROJECTS:
1. Development of hematopoietic precursor cells from human ES cells:
Human embryonic stem (ES) cells provide a powerful model to study human early hematopoiesis. Hematopoietic progenitor cells can be derived either by embryoid body formation or co-culturing with mouse stromal cell S17, OP9 or some other cell lines derived from hematopoietic environment. One main initiative of the lab is to identify primitive hematopoietic cells derived from human ES cells with long-term repopulating and multi-lineage reconstitution ability, as they can potentially be utilized for clinical regenerative therapies to cure various blood diseases.
NOD/SCID mice have been used as a standard model to study the hematopoietic stem cells from umbilical cord blood and bone marrow sources. We have recently found human ES cell-derived cells are capable of long-term hematopoietic engraftment when transplanted into NOD/SCID mice either by intravenous or intra-femur injection.Human CD45+and CD34+cells are identified in the mouse bone marrow 3+ months post-transplantation. To track the engraftment and survival pattern of transplanted cells in vivo, undifferentiated human ES cells that stably transduced with firefly luciferase transgene by lentiviral vector and sleeping beauty transposon system. Non-invasive in vivo bioluminescent imaging has been established in our laboratory to quantitatively evaluate the survival, proliferation and reconstitution mechanism of transplanted undifferentiated and differentiated human ES cells in a real- time manner without need to sacrifice the animal. This system will be useful to more efficiently identify and characterize specific populations of cells derived from human ES cells that are capable of long-term engraftment in vivo.
2 Lymphopoeisis from human ES cells:
The classification of white blood cells is broadly divided into lymphoid and myeloid lineage cells. Human embryonic stem (ES) cells were first used to produce myeloid cells and these protocols are well established. Development of lymphoid lineage cells, such as natural killer (NK) cells, T cells, B cells and dendritic cells, from human ES cell-derived hematopoietic progenitors has been more difficult, but we are making significant progress. Most notably, our lab has developed in vitro protocols to produce NK cells from human ES cells, clearly demonstrating that hESC have lymphoid differentiation potential. human ES cell -derived NK cells express a profile of surface receptors similar to those on primary NK cells. NK cells derived from human ES cell in vitro are also functionally mature. They are able to target and kill tumor cells, both by direct cytolysis and by antibody-dependent cell-mediated cytotoxicity.
Currently, we are testing the ability of human ES cell -derived NK cells to shrink solid tumors established in immunodeficient mice. We can monitor tumor growth in vivo using K562 erythroleukemia cells that ectopically express a luciferase transgene. Without treatment, these tumors grow unchecked. We hypothesize that human ES cell -derived NK cells will curtail tumor growth in vivo and may be able to fully eradicate the cancer cells without harming the host mouse. We are also testing human ES cell -derived NK cell activity against a range of cancer target cells to better define their cytotoxic capabilities and limitations. We are also interested in using hESC to produce T lymphocytes, which are closely related to NK cells.
This system of lymphopoiesis will allow dissection of intracellular gene regulation and extracellular stimuli that regulate development of human lymphocyte populations. There may be key differences in development of mature lymphocytes from hematopoietic progenitor cells derived from human ES cells compared to other sources such as umbilical cord blood or bone marrow. Moreover, the development of human lymphocytes has distinct differences compared to murine systems that have been widely utilized.
3. Application of Bioengineering for more efficient growth and expansion of human ES cell-derived cells.
Human embryonic stem cells have been proposed as a novel cell source for regenerative therapies. Realising their potential for these applications will rely on our capability to propagate these cells into desired functional cell products. This will require two fundamental challenges to be overcome. Expansion from small (milliliter) culture volumes currently in use to larger (liter) scale cultures must be achieved. Secondly, we must better understand and harness the mechanisms that drive stem cells to take on one fate over another. Engineering solutions presented to resolve these issues must yield robust and reproducible end products.
The development of blood cell products presents an attractive first generation of human ES cell-derived therapy. The wealth of knowledge gained from the study of adult human and murine haematopoiesis: population hierarchies, markers and differentiation conditions, provide a solid basis for elucidating the environmental parameters optimal for blood cell formation. In addition, the ease of blood product delivery through transfusion avoids the need for complex multidimensional cell-scaffold constructions and surgical transplantation required by other proposed therapies. Our work aims to develop controlled scaleable culture systems optimized for hematopoietic cell production from human embryonic stem cells.
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