Repopulating hematopoietic stem cells (HSC) are considered to be an ideal cell source for cell and gene therapy of hematological disorders given their capacity to self-renew and differentiate into all myeloid and lymphoid lineages following transplantation. Blood and marrow transplantation (BMT) has been used successfully over the last 3-4 decades to treat malignant blood disorders and genetic disorders of the hematopoietic system. Currently, blood stem cells can be harvested from i) the bone marrow, ii) from peripheral blood after mobilization from the bone marrow or iii) from umbilical cord blood.
To develop more advanced blood cell therapies, including gene therapy, it is essential to understand the molecular regulation of blood stem cells. Scientists at the Division of Molecular Medicine and Gene Therapy study thegeneration of blood stem cells during development, identify novel regulatory pathways that govern stem cell behavior in health and disease, and use these findings to develop new cell and gene therapies for blood disorders.
Specification/Generation of Hematopoietic Stem Cells
During development, hematopoietic stem cells are generated in an area around the aorta. Vascular precursor cells and blood stem cells are believed to have a common origin. After generation de novo, the hematopoietic stem cells move to the fetal liver and self-renew to generate more stem cells. Close to birth the blood stem cells move to the bone marrow where they reside after birth and during adult life. Embryonic stem (ES) cells or induced pluripotent stem cells (iPS cells) are pluripotent stem cells that can generate all tissues of the body. In our department, methods are being developed to generate human hematopoietic stem cells from iPS cells in culture and to investigate the mechanisms that facilitate generation of blood stem cells outside the body (Woods NB et al, Stem Cells, 2011). If successful, the approach can be used to develop safe gene therapies following targeted gene repair in iPS cells.
Regulation of Hematopoietic stem cells
Hematopoietic stem cells are regulated by intrinsic and extrinsic signals. The intrinsic signals represent molecules that govern transcription, epigenetics and the cell cycle. Extrinsic regulators represent growth factors and developmental cues that control signal transduction pathways to determine fate options for hematopoietic cell and progenitor cells. The regulation is very complicated since the different signal transduction pathways interact (integrative signaling) and activate or de-activate intrinsic cellular regulatory factors. The concerted action of these regulatory pathways determines whether the hematopoietic stem cells are quiescent in the bone marrow stem cell niche, keep their HSC properties as they proliferate (self-renewal), commit along the path of differentiation or undergo apoptosis.
In our laboratory, we study known signal transduction pathways and their role in stem cell regulation. The TGF-b family of regulators is of special interest, particularly, TGF-b and BMP. We are studying these signals that are mediated through Smads and those who are non-Smad-mediated. Smad4 is a critical regulator of murine and human hematopoietic stem cells (Karlsson G et al, J Exp Med, 2007; Rörby E et al, Blood, 2012).
We are discovering novel regulators of hematopoiesis through the use of forward genetic screens using lentiviral RNAi and overexpression libraries. Human hematopoietic cells that are transduced with these libraries and are grown in vitro are transplanted into immunocompromized mice to ask whether improved engraftment and self-renewal can be achieved. Sequencing of integrated viral vectors is used to identify expanded clones following bioinformatics analysis. Using this approach p38 signaling was discovered as a critical regulator for human progenitor and stem cells (Baudet A et al, Blood, 2012)
A second approach is to compare dividing and quiescent stem cells using proteomics and metabolomics. Fresh bone marrow stem cells or mobilized peripheral blood stem cells are compared with dividing stem cells (ES cells or fetal liver cells). Recently, Cripto, a developmental cue, has been discovered as a critical regulator for murine hematopoietic stem cells in the stem cell niche and during ex vivo culture (Miharada et al, Cell Stem Cell, 2011). Cripto is activated by hypoxia and mediates its effect through hypoxia-inducing factor-a, whose role in hematopoiesis has also been studied in our department (Rehn M et al, Blood, 2011).
Erythropoiesis: Development of red blood cells
The process of erythropoiesis describes the generation of red blood cells from erythroid progenitors into erythroid precursors (erythroblasts) and finally the development of reticulocytes and mature red blood cells that are required to transport oxygen. Anemia develops when there is a shortage of red blood cells or when they are functionally deficient. In our department, we investigate the molecular mechanisms that regulate erythropoiesis in order to understand the pathogenic mechanisms that lead to anemia. Methods are being developed to increase erythropoiesis and the number of erythroid progenitors to develop mechanism-based therapies for anemia (Flygare J et al, Blood, 2011). Diamond Blackfan anemia is of particular interest since this hypoplastic anemia (shortage of red cell production) that presents in early childhood causes a lot of morbidity and the patients are often transfusion-dependent for a lifetime. Model systems have been developed to investigate the mechanism of this disease in order to develop future therapies for this serious disorder (Jaako P et al, Blood, 2011).
Macrophages and osteoclasts
Monocytes and macrophages are generated from hematopoietic stem cells through intermediate progenitors. They have a role in the non-specific (innate) and specific (adaptive) immune system and can phagocyte and digest cellular debris and pathogens. The most common lysosomal storage disorder, Gaucher disease represents an enzyme deficiency that leads to dysfunctional macrophages through storage of glucosylceramide. Mouse models have been generated to investigate the disease mechanisms and to develop curative gene therapies for this disorder (Enquist IB et al, Proc natl Acad Sci USA, 2006; Enquist IB et al Stem Cells, 2009).
Osteoclasts are “specialized macrophages” that are developed from hematopoietic progenitors. They have an important role in bone resorption. Osteopetrosis is a lethal pediatric illness due to mutations that cause dysfunctional osteoclasts. Mouse models for osteopetrosis have been used to develop curative gene therapies for this disorder and a clinical protocol will be developed to cure this disease (Flores C et al, J Bone Miner Res, 2010).
Malignant blood disorders
Leukemic stem and progenitor cells are studied to unravel the molecular mechanisms of leukemia to allow future design of novel therapies for malignant blood disorders. The studies employ mutant mouse models as well as blood and bone marrow samples from patients to define molecular and cellular defects in leukemia (Quere R et al, Blood, 2011; Reckzeh K et al, Leukemia, 2012).
A key mission of the laboratory and the hematology clinic is to develop novel cell and gene therapies for malignant disorders and genetic defects in hematopoietic cells. To increase the availability of possible stem cell donors for patients with malignant blood disorders we are developing stem cell expansion protocols using mouse models and human umbilical cord blood. Novel therapies for genetic blood disorders are being developed using hematopoietic stem cells from patients and animal models for these disorders. Induced pluripotent stem cells derived from patients are also being used to study disease mechanisms and gene replacement therapies. The efforts in cell and gene therapy focus on the development of permanent cure for genetic disorders of the blood system since the genetically modified cells can be transplanted back to the patient and will be expected to produce genetically corrected cells of all blood lineages.