Marcus O. Muench - US grants

In utero hematopoietic stem cell transplantation (IUT) offers the hope of curing a number of hematological diseases by generating a state of hematopoietic stem cells (HSCs), the levels of donor cell engraftment that can be achieved by IUT are low, limiting the use of this therapy. Our aim is to extend the use of IUT to the treatment of diseases such as sickle cell anemia.. To achieve the high levels of donor cell engraftment needed to treat this disease it is the goal of this proposal to engineer HSCs to have a proliferative advantage over normal HSCs. This proposal will test the hypothesis that introduction of the erythropoietin (EpoR) into HSCs will render these altered cells responsive to erythropoietin (EPO). This will result in the altered HSCs and their progeny having a proliferative advantage over normal progenitors. Truncated forms of EpoR (tEpoR) will also be tested. These tEpoR, having deletions in the negative regulatory region of their cytoplasmic domains, deliver stronger proliferative signals, than EpoR. Fetal HSCs will be used as targets since they offer proliferative advantages over adult cells and are, therefore, susceptible to transduction by retroviral vectors. Lentiviral vectors will also be tested for their capacity to modify fetal as well as postnatal sources of HSCs. The effects of introducing the EpoR genes on the proliferation and differentiation of HSCs will be determined using various in vitro culture systems. It is hypothesized that ectopic expression of either EpoR or tEpoR expression on HSCs can make these cells more competitive than their normal counterparts, modified HSCs will be tested against control HSCs in a mouse model of human fetal hematopoiesis. The in vivo model will also be used to test the effects of EPO administration on the expansion of the modified HSCs. The ability of experiments will further determine if making HSCs responsive to HPO will have detrimental effect on the long-term reconstituting- and multi- lineage potential of HSCs.

DESCRIPTION (adapted from the application) A growing number of hematological diseases can be diagnosed before birth. In some cases, early treatment may benefit the health and survival of the fetus. Either in utero stem cell transplantation (IUT) or fetal gene therapy may treat diseases such as the hemaglobinopathies. This application aims to determine the best method for the introduction of genes into fetal hematopoietic stem cells (HSCs). Fetal HSCs are more proliferative than their adult counterparts and are, therefore, hypothesized to be more susceptible to transduction by retroviral vectors based on murine leukemia virus or human immunodeficiency virus. IUT offers another means of curing a number of hematological diseases by generating a state of hematopoietic chimerism. However, in the absence of any advantage for the donor HSCs, the levels of chimerism that can be achieved by IUT are low. This limits the use of this therapy to very few diseases. Our aim is to extend the use of IUT to the treatment of diseases, such as thalassemia and sickle cell anemia, by engineering HSCs to have a proliferative advantage over normal HSCs. This application will test if introduction of the erythropoietin receptor (EpoR) into HSCs will render these altered cells responsive to erythropoietin (EPO). This will in turn result in the altered HSCs and their progeny having a proliferative advantage over normal progenitors. Truncated forms of EpoR (tEpoR) will also be tested. These tEpoR, having deletions in the negative regulatory region of their cytoplasmic domains, deliver stronger proliferative signals than EpoR. The effects of introducing the EpoR genes on the proliferation and differentiation of HSCs and their progenitor progeny will be determined using various in vitro culture systems. It is hypothesized that ectopic expression of either EpoR or tEpoR will confer the ability of HSCs and early progenitors to proliferate in response to EPO with minimal effect on the differentiation program of these cells. To test if ectopic EpoR or tEpoR expression on HSCs can make these cells more competitive than their normal counterparts, modified HSCs will be tested against control HSCs in a mouse model of human hematopoiesis. The ability of HSCs expressing ectopic EpoR to engraft bone marrow after no or only minimal cytoablation will also be tested. These in vivo experiments will further determine if making HSCs responsive to EPO will have any detrimental effect on the long‑term reconstituting‑ and multilineage‑ potential of HSCs. A positive outcome from the proposed studies would aid in developing treatments for hemoglobinopathies based on generating hematopoietic allochimerism.

[unreadable] DESCRIPTION (provided by applicant): Erythropoiesis dominates hematopoiesis during fetal life to accommodate the rapid expansion in fetal blood volume. Some developmental differences are known to distinguish fetal from adult erythropoiesis such as changes in globin expression and responsiveness to erythropoietin by erythroid progenitors. We hypothesize that a number of unknown genes are differentially regulated between fetal and adult erythroid precursors to accommodate the distinct functions of fetal and adult erythrocytes. Microarray technology is a powerful tool used to compare gene expression between cell populations. To best apply this technology to decipher gene expression during fetal and adult erythropoiesis we have embarked on an effort to improve the tools used to isolate erythroid precursors at different stages of maturation. A panel of single-chain variable fragment antibodies (scFv Abs) was developed, using phage display technology, which recognizes immature nucleated erythrocytes. We aim to determine the molecular identity and expression pattern of the cell-surface markers recognized by these novel reagents. At least three cell surface markers appear to be recognized by the scFv Abs with unique but overlapping expression during erythropoiesis. In addition, to identify the gene products recognized by the scFv Abs we propose to characterize the expression of these cell surface molecules during fetal and adult erythroid development. The second aim of this proposal is to profile gene expression at different stages of fetal and adult erythroid development. This will be accomplished by isolating erythroid precursors at different stages of differentiation from fetal liver and adult bone marrow and then analyzing gene expression using microarray technology. These experiments will both profile the expression of genes during erythroid development and compare expression between adult and fetal erythrocytes. A detailed understanding of gene expression that occurs during the growth and maturation of erythroid progenitors is likely to contribute to improved methods of genetic therapy for hemoglobinopathies. [unreadable] [unreadable]

PROJECT SUMMARY (See instructions): The purpose of Core C, the Cell Transplantation and Analysis Core, is to provide critical support for cell sourcing, isolation and analysis needs of research performed under the three Projects and Core B, The specific service aims of Core C are: Aim 1: To procure specific cell types and tissues from human embryonic, fetal, neonatal and adult tissue specimens. The requirements of project scientists for primary human tissue specimens such as skin, hematopoietic and mesenchymal cells will be met by obtaining these tissues from fetal, neonatal and adult specimens. Aim 2: To provide flow cytometric analysis and sorting services to project scientists. Expertise and assistance with flow cytometric analysis and fluorescence-activated cell sorting (FACS) will be provided to project scientists. Aim 3: To assay teratoma formation in immunodeficient mice. One hallmark of induced pluripotent stem (IPS) cells is their capacity to form teratomas when transplanted into immunodeficient mice. IPS cells and differentiated cells generated by the 3 projects will be assayed for teratoma forming ability to measure pluripotentiality. Aim 4: To assay long-term multilineage hematopoietic reconstitution of candidate hematopoietic stem cells (HSCs) generated from IPS cells. The capacity of hematopoietic cells created from IPS cells under Project 3 to reconstitute erythroid, myeloid and lymphoid lineages for at least 12 weeks will be tested by transplantation into immunodeficient mice. The work will be performed under the guidance of Dr. Marcus Muench at Blood systems Research Institute utilizing the Core Immunology and Cell, Tissue and Vivarium Cores already established at that institution.

PROJECT SUMMARY (See instructions): The overall goal of Project 3 is to develop a method to efficiently and reproducibly differentiate induced pluripotent stem (IPS) cells into hematopoietic stem cells (HSCs). Aim 1 of this project will determine the best conditions for the mesodermal differentiation of iPS cells and the generation of HSCs. This aim will test the hypothesis that an engineered human stromal cell line offers the best method to reliably differentiate IPS cells into hematopoietic precursors. Experiments will compare methods of differentiating IPS cells using embryoid bodies (EB) cultures and stromal cell lines to support the differentiation of IPS cells into HSCs. Various elements of these two differentiation methods will be studied to determine the best method to promote mesodermal differentiation and HSC creation. The second aim is to determine the optimal cytokine conditions for the survival, growth and expansion of HSCs generated from iPS cells. This aim will test the hypothesis that HSCs generated from IPS are more similar in their cytokine responses to fetal HSCs than adult HSCs. Accumulation of HSCs in culture requires conditions that favor their survival and minimize their differentiation into committed progenitors. Various cytokines known to play a role in the early stages of hematopoiesis will be tested in combination to compare the similarity of HSCs generated from iPS cells to those isolated from fetal tissues, umbilical cord blood and adult peripheral blood. These experiments will optimize culture conditions for the production of HSCs. The third aim will determine the variability in the capacity of different IPS cell lines to differentiate into HSCs. This aim will test the hypothesis that different iPS cells lines are similarly capable to form HSCs that can generate long-term multilineage reconstitution in immunodeficient mice. This aim will test isolated HSCs derived from multiple IPS cell lines for their capacity to provide long-term reconstitution without teratoma or leukemia formation, chromosomal abnormalities or other obvious functional deficiencies. The outcome of the experiments will be development of technology to efficiently generate transplantable HSCs from genetically corrected IPS cells.