My laboratory seeks a better understanding of the biology, pathology, and clinical utility of hematopoietic stem cells and an understanding of the role of various tissue stem cells in disease and development. We focus on pluripotent stem cells, murine and human embryology, blood development, and drug therapy for chronic myeloid leukemia. Our ultimate goals are to improve drug and transplantation therapies for patients with malignant and genetic bone marrow disease and to define fundamental principles of how stem cells contribute to tissue regeneration and repair.
For the majority of hematopoietic malignancies for which no targeted therapy exists, and for patients with genetic bone marrow conditions such as immune deficiency and bone marrow failure, hematopoietic stem cell transplantation remains the best hope for a cure. Unfortunately, the toxicity of current regimens and the shortage of suitable donors limit more widespread use. We endeavor to develop hematopoietic stem cells from pluripotent human stem cells as a platform for combining gene repair and cell replacement therapy.
Pluripotent stem cells can generate any of the hundreds of distinct cell types in the body; thus they are valuable tools for studying tissue formation and may one day provide a resource for cellular therapies. A major goal of our program is to derive customized pluripotent stem cells from patients with specific diseases, chiefly those affecting hematopoiesis. In murine systems, we have generated embryonic stem (ES) cells by nuclear transfer and parthenogenesis and modeled transplantation therapy to treat genetic immunodeficiency and hemoglobin disorders such as thalassemia and sickle cell anemia. We are using RNA interference knockdown methods to model distinct bone marrow failure disorders in human ES cells. We are exploring strategies to enhance the efficiency of human ES cell derivation from embryos created by in vitro fertilization, and to generate customized human ES cells by nuclear transfer.
To model human disease, we are exploiting the reprogramming of human somatic cells to pluripotency with defined factors. By transducing human fibroblasts with three or four transcription factors (Oct4, Sox2, and Myc or KLF4 or both), we can derive cell lines that exhibit the cardinal features of human ES cells: immortal self-renewal, multilineage differentiation potential in vitro, and teratoma formation in immune-deficient mice—currently the gold standard definition of human stem cell pluripotency.
We have generated a variety of these induced pluripotent stem (iPS) cell lines from patients with bone marrow failure syndromes, and we are comparing the behavior of these lines with that of human ES cells that model gene deficiency states by RNA interference. Moreover, we have created iPS lines from patients with a variety of single-gene or complex genetic disorders, such as muscular dystrophy, Huntington's disease, Down syndrome, and diabetes. We have developed methods for producing transgene-free iPS cells that may be suitable for future applications in clinical transplantation.
Our overall goals are to generate autologous stem cells from patients with genetic bone marrow disease, repair gene defects in vitro, direct pluripotent stem cells into hematopoietic stem cells, and engraft patients in a combined platform of gene therapy and stem cell transplantation (Figure).
One of our long-standing interests is how hematopoietic stem cells develop in the vertebrate embryo. We have explored several experimental systems to identify principles that could help direct the formation of hematopoietic tissues from pluripotent cell types in vitro. By transducing hematopoietic progenitors with hoxb4, a homeodomain-containing transcription factor that enhances hematopoietic stem cell cycling and competitive engraftment, we can engraft mice with hematopoietic stem cells derived from murine ES cells. Although the cells show the cardinal features of self-renewal and multilineage (lymphoid-myeloid) differentiation potential in vivo, the cells are not the precise equivalents of bone marrow–derived hematopoietic stem cells. Presumably, we lack critical signals within our in vitro differentiation system to faithfully mimic embryonic blood formation.
With our collaborator Leonard Zon (HHMI, Harvard Medical School), we have explored the role of the cdx-hox pathway in regulating blood development and have identified morphogen pathways that activate cdx and hox genes and promote blood formation within zebrafish and murine embryos and in cultures of differentiating murine and human ES cells. We are investigating how biomechanical forces influence the developmental maturation of the definitive hematopoietic system of mammals.
In studying chronic myeloid leukemia, the classic malignant condition of hematopoietic stem cells, we use pathology to illustrate biology. This disease is caused by an activated BCR/ABL tyrosine kinase. Because of its origins in stem cells, this type of leukemia is poorly responsive to standard induction chemotherapy. Until recently, hematopoietic stem cell replacement by bone marrow transplantation was the only cure, but ABL kinase blockade by Gleevec (imatinib) has proven remarkably effective. Although Gleevec induces durable remissions in virtually all chronic-phase patients, the leukemia is eradicated in only a small percentage (as determined by sensitive polymerase chain reaction methods), and relapse rates of up to 4 percent per year imply that residual leukemia persists. Relapse is particularly problematic for patients treated in blast crisis and is typically accompanied by ABL kinase domain mutations that thwart drug binding. The most vexing mutation is an isoleucine substitution at the "gatekeeper" threonine residue within the ATP-binding site (T315I).
Our program in leukemia research aims to elucidate mechanisms of resistance to ABL kinase inhibitors, characterize drugs that are active against T315I, and develop methods for mutation detection to guide patient therapy.
We have recently demonstrated that the protein Lin28 can modulate the processing of the let7 family of tumor-suppressor microRNAs. In exploring the relationship between Lin28 and tumorigenesis, we have discovered that Lin28 is aberrantly expressed in a subset of human tumors, including hepatocellular carcinoma, ovarian cancer, Wilm's tumor, germ cell tumors, and the blast crisis stage of chronic myeloid leukemia. Ironically, Lin28 also functions as a reprogramming factor for human somatic cells, and thus our dual efforts at investigating pluripotency and malignancy have found a natural topic of intersection, perhaps reflecting the fact that somatic cell reprogramming and tumorigenesis share common pathways driving dedifferentiation.