Xiaomiao Li

The role of the donor in the repair of the marrow vascular niche following hematopoietic stem cell transplant.

Bone marrow sinusoids maintain homeostasis between developing hematopoietic cells and the circulation, and they provide niches for hematopoietic progenitors. Sinusoids are damaged by chemotherapy and radiation. Hematopoietic stem cells (HSCs) have been shown to produce endothelial progenitor cells that contribute to the repair of damaged blood vessels. Because HSCs home to the marrow during bone marrow transplant, these cells may play a role in repair of marrow sinusoids. Here, we explore the role of donor HSCs in the repair of damaged sinusoids following hematopoietic stem cell transplant. We used three methods to test this role: (a) expression of platelet endothelial cell adhesion molecule to identify endothelial progenitors and the presence of the Y chromosome to identify male donor cells in female recipients; (b) presence of the Y chromosome to identify male donor cells in female recipients, and expression of the panendothelial marker mouse endothelial cell antigen‐32 to identify sinusoidal endothelium; and (c) use of Tie‐2/green fluorescent protein mice as donors or recipients and presence of Dil‐Ac‐LDL to identify sinusoids. We found that sinusoids were predominantly host‐derived posttransplant. Donor cells spread along the marrow vasculature early post‐transplant in a pattern that matched stromal‐derived factor‐1 expression. Furthermore, these engrafting progenitors were positioned to provide physical support, as well as growth and survival signals in the form of vascular‐endothelial growth factor‐A. Occasionally, donor cells provide cellular “patches” in the damaged sinusoids, although this occurred at a low level compared with hematopoietic engraftment. Donor support for the repair of the marrow vascular niche may be a critical first step of hematopoietic engraftment.

Bone marrow sinusoidal endothelial cells undergo nonapoptotic cell death and are replaced by proliferating sinusoidal cells in situ to maintain the vascular niche following lethal irradiation

OBJECTIVE Bone marrow sinusoids remain predominantly host-derived following bone marrow transplantation. Systematic analysis was conducted at the cellular level to investigate how the host sinusoidal structures survived after lethal irradiation. MATERIALS AND METHODS Apoptosis and cell proliferation assays were performed on bone marrow sections at various time points during the first 2 weeks postirradiation to study the extent of damage to sinusoidal endothelial cells from lethal irradiation and to determine whether cell proliferation contributes to the recovery of the sinusoidal system. RESULTS Phosphorylated H2AX was present in both hematopoietic and sinusoidal endothelial cells 3 hours after irradiation demonstrating DNA damage. Three days after irradiation, some sinusoidal endothelial cells became terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling -positive, but were caspase-3 and in situ oligo ligation -negative, suggesting nonapoptotic DNA fragmentation. Clusters of sinusoidal endothelial cells that expressed Ki67 appeared 3 days after irradiation, and increased through day 7. These Ki67-positive endothelial cells were host-derived. Bromodeoxyuridine-positive endothelial cells were present in the Ki67-positive areas confirming endothelial cell replication. Twenty percent of the sinusoidal endothelial cells were lost by day 3 after irradiation. The total number of endothelial cells remained relatively unchanged between day 3 and day 14. These results demonstrate that lethal irradiation resulted in limited, nonapoptotic sinusoidal endothelial cell loss, followed by proliferation of preexisting host-derived mature sinusoidal endothelial cells. Our data suggest that DNA repair mechanisms and proliferation of host endothelial cells within the sinusoids are involved in maintenance of the structural integrity of the bone marrow vascular niche following lethal irradiation.

Conditional Deletion of Jak2 Reveals an Essential Role in Hematopoiesis throughout Mouse Ontogeny: Implications for Jak2 Inhibition in Humans

Germline deletion of Jak2 in mice results in embryonic lethality at E12.5 due to impaired hematopoiesis. However, the role that Jak2 might play in late gestation and postnatal life is unknown. To understand this, we utilized a conditional knockout approach that allowed for the deletion of Jak2 at various stages of prenatal and postnatal life. Specifically, Jak2 was deleted beginning at either mid/late gestation (E12.5), at postnatal day 4 (PN4), or at ∼2 months of age. Deletion of Jak2 beginning at E12.5 resulted in embryonic death characterized by a lack of hematopoiesis. Deletion beginning at PN4 was also lethal due to a lack of erythropoiesis. Deletion of Jak2 in young adults was characterized by blood cytopenias, abnormal erythrocyte morphology, decreased marrow hematopoietic potential, and splenic atrophy. However, death was observed in only 20% of the mutants. Further analysis of these mice suggested that the increased survivability was due to an incomplete deletion of Jak2 and subsequent re-population of Jak2 expressing cells, as conditional deletion in mice having one floxed Jak2 allele and one null allele resulted in a more severe phenotype and subsequent death of all animals. We found that the deletion of Jak2 in the young adults had a differential effect on hematopoietic lineages; specifically, conditional Jak2 deletion in young adults severely impaired erythropoiesis and thrombopoiesis, modestly affected granulopoiesis and monocytopoiesis, and had no effect on lymphopoiesis. Interestingly, while the hematopoietic organs of these mutant animals were severely affected by the deletion of Jak2, we found that the hearts, kidneys, lungs, and brains of these same mice were histologically normal. From this, we conclude that Jak2 plays an essential and non-redundant role in hematopoiesis during both prenatal and postnatal life and this has direct implications regarding the inhibition of Jak2 in humans.

Stable Integration of Recombinant Adeno-Associated Virus Vector Genomes After Transduction of Murine Hematopoietic Stem Cells

We previously reported that among single-stranded adeno-associated virus (ssAAV) vectors, serotypes 1 through 5, ssAAV1 is the most efficient in transducing murine hematopoietic stem cells (HSCs), but viral second-strand DNA synthesis remains a rate-limiting step. Subsequently, using double-stranded, self-complementary AAV (scAAV) vectors, serotypes 7 through 10, we observed that scAAV7 vectors also transduce murine HSCs efficiently. In the present study, we used scAAV1 and scAAV7 shuttle vectors to transduce HSCs in a murine bone marrow serial transplant model in vivo, which allowed examination of the AAV proviral integration pattern in the mouse genome, as well as recovery and nucleotide sequence analyses of AAV-HSC DNA junction fragments. The proviral genomes were stably integrated, and integration sites were localized to different mouse chromosomes. None of the integration sites was found to be in a transcribed gene, or near a cellular oncogene. None of the animals, monitored for up to 1 year, exhibited pathological abnormalities. Thus, AAV proviral integration-induced risk of oncogenesis was not found in our study, which provides functional confirmation of stable transduction of self-renewing multipotential HSCs by scAAV vectors as well as promise for the use of these vectors in the potential treatment of disorders of the hematopoietic system.

High Levels of Acetylated Low-Density Lipoprotein Uptake and Low Tyrosine Kinase With Immunoglobulin and Epidermal Growth Factor Homology Domains-2 (Tie2) Promoter Activity Distinguish Sinusoids From Other Vessel Types in Murine Bone Marrow

Background— The bone marrow contains a variety of blood vessels that have different functions in bone marrow maintenance and hematopoiesis. Arterioles control the flow of blood into bone marrow compartments, and sinusoids serve as a conduit to the bloodstream and as niches for megakaryocyte development. Most studies of bone marrow vasculature, including studies quantifying changes in the marrow vascular by microvascular density, do not differentiate between different types of marrow vessels. Recognizing changes in different types of blood vessels after chemotherapy exposure or during leukemia development has important physiological implications. We hypothesized that the functional heterogeneity of marrow vasculature could be recognized through the use of functional markers such as tyrosine kinase with immunoglobulin and epidermal growth factor homology domains-2 (Tie2) expression or 1,1′-dioctadecyl -3,3,3′,3′-tetramethyl-indocarbocyanine perchlorate with acetylated low-density lipoprotein (DiI-Ac-LDL) uptake. Methods and Results— When transgenic mice with green fluorescent protein (GFP) expressed downstream of the Tie2 promoter were injected with Ac-LDL, Ac-LDL was specifically endocytosed by sinusoids, and Tie2 expression was more pronounced in the arteries, arterioles, and transitional capillaries. Combining these 2 functional endothelial markers and using confocal microscopy to obtain 3-dimensional images, we identified transitional zones where arterioles emptied into the sinusoids. Alternatively, coinjection of lectin with DiI-Ac-LDL has a similar result in normal mice, as seen in Tie2/GFP mice, and can be used to differentiate vessel types in nontransgenic mice. Conclusions— These results demonstrate that bone marrow vasculature is functionally heterogeneous. Methods to study changes in the marrow vasculature using microvascular density or quantifying changes in the vascular niche need to take this heterogeneity into account.

High-Efficiency Transduction of Primary Human Hematopoietic Stem Cells and Erythroid Lineage-Restricted Expression by Optimized AAV6 Serotype Vectors In Vitro and in a Murine Xenograft Model In Vivo

We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34+ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34+ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.

Optimizing the transduction efficiency of capsid-modified AAV6 serotype vectors in primary human hematopoietic stem cells in vitro and in a xenograft mouse model in vivo

BACKGROUND AIMS Although recombinant adeno-associated virus serotype 2 (AAV2) vectors have gained attention because of their safety and efficacy in numerous phase I/II clinical trials, their transduction efficiency in hematopoietic stem cells (HSCs) has been reported to be low. Only a few additional AAV serotype vectors have been evaluated, and comparative analyses of their transduction efficiency in HSCs from different species have not been performed. METHODS We evaluated the transduction efficiency of all available AAV serotype vectors (AAV1 through AAV10) in primary mouse, cynomolgus monkey and human HSCs. The transduction efficiency of the optimized AAV vectors was also evaluated in human HSCs in a murine xenograft model in vivo. RESULTS We observed that although there are only six amino acid differences between AAV1 and AAV6, AAV1, but not AAV6, transduced mouse HSCs well, whereas AAV6, but not AAV1, transduced human HSCs well. None of the 10 serotypes transduced cynomolgus monkey HSCs in vitro. We also evaluated the transduction efficiency of AAV6 vectors containing mutations in surface-exposed tyrosine residues. We observed that tyrosine (Y) to phenylalanine (F) point mutations in residues 445, 705 and 731 led to a significant increase in transgene expression in human HSCs in vitro and in a mouse xenograft model in vivo. CONCLUSIONS These studies suggest that the tyrosine-mutant AAV6 serotype vectors are the most promising vectors for transducing human HSCs and that it is possible to increase further the transduction efficiency of these vectors for their potential use in HSC-based gene therapy in humans.

Recombinant Self-Complementary Adeno-Associated Virus Serotype Vector-Mediated Hematopoietic Stem Cell Transduction and Lineage-Restricted, Long-Term Transgene Expression in a Murine Serial Bone Marrow Transplantation Model

Although conventional recombinant single-stranded adeno-associated virus serotype 2 (ssAAV2) vectors have been shown to efficiently transduce numerous cells and tissues such as brain and muscle, their ability to transduce primary hematopoietic stem cells (HSCs) has been reported to be controversial. We have previously documented that among the ssAAV serotype 1 through 5 vectors, ssAAV1 vectors are more efficient in transducing primary murine HSCs, but that viral second-strand DNA synthesis continues to be a rate-limiting step. In the present studies, we evaluated the transduction efficiency of several novel serotype vectors (AAV1, AAV7, AAV8, and AAV10) and documented efficient transduction of HSCs in a murine serial bone marrow transplantation model. Self-complementary AAV (scAAV) vectors were found to be more efficient than ssAAV vectors, and the use of hematopoietic cell-specific enhancers/promoters, such as the human beta-globin gene DNase I-hypersensitive site 2 enhancer and promoter (HS2-betap) from the beta-globin locus control region (LCR), and the human parvovirus B19 promoter at map unit 6 (B19p6), allowed sustained transgene expression in an erythroid lineage-restricted manner in both primary and secondary transplant recipient mice. The proviral AAV genomes were stably integrated into progenitor cell chromosomal DNA, and did not lead to any overt hematological abnormalities in mice. These studies demonstrate the feasibility of the use of novel scAAV vectors for achieving high-efficiency transduction of HSCs as well as erythroid lineage-restricted expression of a therapeutic gene for the potential gene therapy of beta-thalassemia and sickle cell disease.

Optimization of Recombinant Adeno-Associated Viral Vectors for Human β-Globin Gene Transfer and Transgene Expression

Therapeutic levels of expression of the beta-globin gene have been difficult to achieve with conventional retroviral vectors without the inclusion of DNase I-hypersensitive site (HS2, HS3, and HS4) enhancer elements. We generated recombinant adeno-associated viral (AAV) vectors carrying an antisickling human beta-globin gene under the control of either the beta-globin gene promoter/enhancer or the erythroid cell-specific human parvovirus B19 promoter at map unit 6 (B19p6) without any enhancer, and tested their efficacy in a human erythroid cell line (K-562) and in primary murine hematopoietic progenitor cells (c-kit(+)lin()). We report here that (1) self-complementary AAV serotype 2 (scAAV2)-beta-globin vectors containing only the HS2 enhancer are more efficient than single-stranded AAV (ssAAV2)-beta-globin vectors containing the HS2+HS3+HS4 enhancers; (2) scAAV2-beta-globin vectors recombine with scAAV2-HS2+HS3+HS4 vectors after dual-vector transduction, leading to transgene expression; (3) scAAV2-beta-globin as well as scAAV1-beta-globin vectors containing the B19p6 promoter without the HS2 enhancer element are more efficient than their counterparts containing the HS2 enhancer/beta-globin promoter; and (4) scAAV2-B19p6-beta-globin vectors in K-562 cells, and scAAV1-B19p6-beta-globin vectors in murine c-kit(+)lin() cells, yield efficient expression of the beta-globin protein. Thus, the combined use of scAAV vectors and the parvovirus B19 promoter may lead to expression of therapeutic levels the beta-globin gene in human erythroid cells, which has implications in the use of these vectors in gene therapy of beta-thalassemia and sickle cell disease.

Molecular mechanisms of platelet and stem cell rebound after 5-fluorouracil treatment.

Sublethal irradiation and 5-fluorouracil (5-FU) treatment are two commonly used myelosuppressive methods used in the study of hematopoiesis. These methods have been considered interchangeable by some researchers because the morphological changes in the bone marrow to these treatments are similar. Here, we sought to compare the responses of hematopoietic cells, stem and progenitor cells and the bone marrow microenvironment to these treatments. Although bone marrow cellularity decreased after both treatments, the underlying mechanism of the bone marrow cell regression and recovery were very different between the two models. We found: 1. Myeloid cells and lymphoid cells had different sensitivity to the different treatments. 2. Following an initial decrease in stem cell number, 5-FU treated mice had profound thrombopoietin (Tpo) dependent stem cell rebound above baseline levels. 3. Platelet rebound in 5-FU treated animals was not the result of stem cell rebound. 4. Stem cell and platelet rebound did not occur in sub-lethally irradiated mice. 5. Platelet rebound resulted from an indirect effect of 5-FU on the microenvironment cells, but not a direct effect on the stem cells. 6. Microarray studies demonstrated that up-regulation of the angiopoietin-1/Tie2 signaling pathway coincided with platelet rebound. 7. Suppression of genes involved in chromosomal organization coincided with stem cell and platelet rebound.