Hematopoietic stem cells reside within the bone marrow post-natally, providing all hematopoietic lineages for the life of the host, and their extensive characterization over the last several decades has led to clinical application including bone marrow transplantation for benign and malignant disorders affecting the hematopoietic compartment. Isolation of bone marrow cells based upon expression of the CD34 antigen enriches for the primitive compartment, and techniques to isolate the CD34-positive population are commonly used in clinical practice. Assays for true hematopoietic stem cells require transplantation and repopulation, and thus studies querying such potential among human cells are difficult. We have thus developed a xenograft model with robust human hematopoietic stem cell engraftment, and have demonstrated engraftment of all cell lineages. This assay will allow us to test globin vectors for the first time among engrafted human cells in a relevant model. Recently, a method for the isolation of murine bone marrow cells capable of reconstituting hematopoiesis after lethal irradiation based solely upon dual wavelength flow cytometry after staining with the vital dye, Hoechst 33342, was described by Goodell et. al. The molecular basis of these side population cells was recently attributed to expression of the ABC transporter, ABCG2. Side population (SP) cells are highly enriched for hematopoietic repopulating ability, with one cell capable of reconstituting hematopoiesis in irradiated recipient mice. We reasoned that this phenotype is conserved among organs with the capacity for post-natal regeneration. We have recently isolated SP cells from the non-parynchymal portion of human cadaveric liver and have cultured these cells in hepatic culture media. Hepatic SP cells generated colonies of cells demonstrating granule rich cytoplasm and dense, often double nuclei consistent with hepatocytes. These hepatocyte-like cells expressed markers of human hepatocytes including HepPar, cytokeratin-8, and human albumin. RT-PCR confirmed the expression of hepatocyte markers including albumin, cytokeratin-18, along with the more specific markers, alpha-1-antitrypsin and the human P450 gene, CYP2B6. We have now developed an in vivo rescue model in NOD/SCID mice treated with sublethal dosing of carbon-tetra-chloride, and initial results suggest that SP cells are capable of affecting a normalization of liver function test similar to mature hepatocytes when compared to controls. Further in vivo characterization of these SP cells is ongoing. Additionally, SP cells have been isolated from the adult pancreas, and microarray analysis demonstrates gene expression profile similar to that of bone marrow derived SP cells. Initial attempts to differentiate pancreatic SP cells toward a mature beta-cell phenotype have met with only limited success and these studies are ongoing, yet their quiescence in culture raises the question whether beta-cells can regenerate in adult hosts. We have thus developed a method for assessing beta cell regeneration post-natally using 14C dating by accelerator mass spectrometry. Initial results suggest limited turnover. Further, samples from individuals who received the DNA labeling agents BrdU and IdU were obtained and analyzed for evidence of turnover. These samples also suggest limited turnover of adult beta cells. We have thus turned back to animal models to further query the potential for tolerance induction in Type I diabetes mellitus, as this tolerance will be necessary in a number of disease states, including our main focus, allogeneic hematopoietic stem cell transplantation. Studies are ongoing utilizing rapamycin, an agent we have demonstrated to achieve operational tolerance in animal models and humans, to determine if this agent can lead to antigen specific tolerance in a naturally occurring model of Type I dieabetes in the mouse. This work will be used to support collaborative studies with clinical investigators in the field.

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Leonard, Alexis; Tisdale, John F (2018) Stem cell transplantation in sickle cell disease: therapeutic potential and challenges faced. Expert Rev Hematol 11:547-565
Uchida, Naoya; Haro-Mora, Juan J; Fujita, Atsushi et al. (2017) Efficient Generation of ?-Globin-Expressing Erythroid Cells Using Stromal Cell-Derived Induced Pluripotent Stem Cells from Patients with Sickle Cell Disease. Stem Cells 35:586-596
Uchida, Naoya; Fujita, Atsushi; Hsieh, Matthew M et al. (2017) Bone Marrow as a Hematopoietic Stem Cell Source for Gene Therapy in Sickle Cell Disease: Evidence from Rhesus and SCD Patients. Hum Gene Ther Clin Dev :
Fujita, Atsushi; Uchida, Naoya; Haro-Mora, Juan J et al. (2016) ?-Globin-Expressing Definitive Erythroid Progenitor Cells Generated from Embryonic and Induced Pluripotent Stem Cell-Derived Sacs. Stem Cells 34:1541-52
Uchida, Naoya; Green, Rashidah; Ballantine, Josiah et al. (2016) Kinetics of lentiviral vector transduction in human CD34(+) cells. Exp Hematol 44:106-15
Evans, Molly E; Kumkhaek, Chutima; Hsieh, Matthew M et al. (2014) TRIM5? variations influence transduction efficiency with lentiviral vectors in both human and rhesus CD34(+) cells in vitro and in vivo. Mol Ther 22:348-58
Kovtunovych, Gennadiy; Ghosh, Manik C; Ollivierre, Wade et al. (2014) Wild-type macrophages reverse disease in heme oxygenase 1-deficient mice. Blood 124:1522-30
Herman, S E M; Sun, X; McAuley, E M et al. (2013) Modeling tumor-host interactions of chronic lymphocytic leukemia in xenografted mice to study tumor biology and evaluate targeted therapy. Leukemia 27:2311-21
Kumkhaek, Chutima; Aerbajinai, Wulin; Liu, Wenli et al. (2013) MASL1 induces erythroid differentiation in human erythropoietin-dependent CD34+ cells through the Raf/MEK/ERK pathway. Blood 121:3216-27
Perl, Shira; Perlman, Jordan; Weitzel, R P et al. (2013) Addition of rapamycin to anti-CD3 antibody improves long-term glycaemia control in diabetic NOD mice. PLoS One 8:e67189

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