ALBERT

Description: CD34 negative hematopoietic stem cells (CD34− HSCs) were identified in mice and humans. Human HSCs are evaluated as severe combined immunodeficient mouse (SCID)-repopulating cells (SRCs), originally identified by the ability to reconstitute hematopoiesis in nonobese diabetic (NOD)/SCID mouse. CD34− cord blood (CB) cells have been hard to engraft in NOD/SCID mice until recent report of successlul engraftment by intra-bone marrow transplantation (iBMT). However, CD34− bone marrow (BM) cells have not been analyzed precisely. We prepared lineage negative CD34 negative (Lin-CD34−) cells by negative selection using CD2,3,7,14,16,19,20,33,34,36,41,56,127, and GlyA antibody. Lin-CD34− BM cells did not engraft in NOD/SCID mice even by using iBMT (0/6). In the previous study, we reported that Lin-CD34− BM cells were able to differentiate into CD34+ cells accompanied by the emergence of colony forming activity after 7 days of stroma-dependent culture, while SRC activity was not detected. (BMT 28, 587–595, 2001) Here we cultured Lin-CD34− BM cells on stroma cells transfected with human angiopoietin-1 cDNA (AHESS-5), since we detected Tie-2 expression on Lin-CD34− BM cells. AHESS-5 supported induction of CD34 much better than HESS-5 cells or empty vector transfected control cells (EVHESS-5), and the effect was blocked by anti-Tie-2 antibody (Fig.1). Furtheremore, CD34+ cells produced from CD34− BM cells engrafted in NOD/SCID mice (11/12). As previously reported, CD34− CB cells differentiate CD34+ cells and acquire SRC activity by stroma-dependent culture without angiopoietin-1. These results highlighted the characteristic differences of CD34− HSCs of BM from CB and the unique role of BM niche for CD34− HSCs. Fig. 1 CD34 expression on Lin − CD34 − BM cells after 7 days of culture Fig. 1. CD34 expression on Lin−CD34− BM cells after 7 days of culture

Description: The SCID-repopulating cell (SRC) pool is shown to be heterogeneous and is composed of at least two distinct subsets; short-term and long-term repopulating cells (STRCs and LTRCs), which appear in different time points following transplantation. However, the precise characteristics and their relationships regarding the stem cell function remain elusive. To clarify the specific stem cell activity of each SRC clones that contribute to various stages of hematopoietic reconstitution, we examined the functional aspects of individual SRCs. To determine the repopulating dynamics of individual SRC clones in vivo, we traced the kinetics of individual SRC clones by LAM-PCR based virus integration site analysis. Individual SRC clones which repopulate in each NOG mouse that received EGFP-transduced fractionated CD34+ populations were analyzed at two time points. At 3 weeks after transplantation, BM cells were aspirated from tibia of each recipient, and at 18 weeks recipients were sacrificed and BM cells were recovered from 4 long bones. At each time point, EGFP-expressing human hematopoietic lineage cells were sorted for integration site analysis by LAM-PCR, and the fate of individual SRC clones in the same recipient was examined by clone-tracking analysis. Using primers that were designated based on the genomic sequence information of the CD33+ myeloid cell integration site, we clonally traced distribution of each clone in lineage cells; CD34+ stem/progenitor, T-, and B-lymphoid cells. We found that the early phase of hematopoietic reconstitution was attributed to transient myeloid-restricted clones which rapidly exhausted from the CD34+ stem cell pool. Interestingly, the multilineage cell-producing clones that were responsible for the later phase of hematopoiesis were distinct from the transient myeloid-restricted clones, and these clones continuously self-replicated in the CD34+ stem cell pool. Next, CD34+ cells from the primary recipients were divided into two secondary recipients, and the fate of individual SRC clones in different phases was traced using the paired secondary mice. One recipient was sacrificed at 3 weeks, and the other recipient was sacrificed at 18 weeks after secondary transplantation. First, clones that were detected at the early phase in one recipient were also detected at the later phase in the other recipient (80%). This is clonal evidence that LTRC in the primary recipient produces STRC as well as self-replicating secondary transplantable LTRC. Second, all clones in the secondary recipients were also detected in the primary donor; however, most of clones (68.3%) found in the primary recipients did not contribute to the secondary recipient. In addition, LTRC clones detected in the CD34+ stem cell pool of secondary recipient demonstrated much larger clone size compared to primary recipient. These indicated that the quiescent LTRC clones in the primary recipients were stimulated by transplantation, there by expanded clonally in the secondary recipient and contributed to the later phase of hematopoiesis. Our clonal tracking study clearly demonstrated that the hierarchical structure of the human HSC pool composed of distinct clonal subsets which were heterogeneous in the self-renewal capacity and differentiation ability.

Description: Acute promyelocytic leukemia (APL) is a subtype of acute myeloid leukemia (AML) characterized by the formation of a PML-RARa fusion protein, which leads to the accumulation of abnormal promyelocytes. Xenograft mouse models with human leukemic cells have advantages for analyzing the human leukemias in vivo, especially for genetic analyses. However, human primary APL cells are difficult to engraft even in very severely immunodeficient mice, such as NOD/shi-SCID IL2Rg-/- (NOG) mice. In order to understand the mechanisms involved in human APL leukemogenesis, we established a humanized in vivo APL model using the transplantation of PML-RARA-transduced CD34+ cells from human cord blood into NOG mice. The expression of PML-RARa in the CD34+ cells disrupted the nuclear bodies in vitro. The clonogenic assay showed that PML-RARa inhibited the total colony formation, but favored the growth of myeloid colonies. When CD34+ cells with PML-RARA were transplanted, they proliferated in the NOG mice for more than three to four months after transplantation (in 24 out of the 34 mice). All 16 mice with more than 3,000 PML-RARA-transduced CD34+ cells were engrafted, while the engraftment was only detected in eight out of 18 mice when the cell density used for transplantation was less than 3,000 cells. These cells possessed abundant azurophilic abnormal granules in the cytoplasm, and some of them had bundles of Auer rods. They expressed CD13, CD33 and CD117, but not HLA-DR or CD34. In addition, the gene expression analysis revealed that these cells and human primary APL were clustered together among various types of AML, suggesting that these induced APL cells well recapitulated human primary APL. Similar to human primary APL, the induced APL cells possessed the ability for myeloid differentiation after treatment with all-trans retinoic acid in vitro and in vivo, and a very low potential for re-transplantation, which was similarly observed in both unsorted induced APL cells and the CD34- fraction. When human cord blood was fractionated before the PML-RARA transduction, the CD34+/CD38+ cells and common myeloid progenitors (CMP) in the CD34+/CD38+ cells led to the efficient development of APL in vivo. These findings demonstrate that CMP is a target for PML-RARA in APL, whereas the resultant CD34- APL cells may share the ability to maintain the leukemia. Disclosures No relevant conflicts of interest to declare.

Description: To characterize human hematopoietic stem cells (HSCs), xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSCs, LDA is a statistical method and does not directly establish that a single HSC has self-renewed in vitro. This would require a direct clonal method and has not been done. By using lentiviral gene marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. Of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than 2 mice after serum-free and stroma-dependent culture. Some of the clones were secondary transplantable. This indicates symmetric self-renewal divisions in vitro. On the other hand, 54 clones (73%) present in only 1 mouse may result from asymmetric divisions in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.

Description: Stem cells of highly regenerative organs including blood are susceptible to endogenous DNA damage caused by both intrinsic and extrinsic stress. Response mechanisms to such stress equipped in hematopoietic stem cells (HSCs) are crucial in sustaining hematopoietic homeostasis but remain largely unknown. In this study, we demonstrate that serial transplantation of human HSCs into immunodeficient mice triggers replication stress that induces incremental elevation of intracellular reactive oxygen species (ROS) levels and the accumulation of persistent DNA damage within the human HSCs. This accumulation of DNA damage is also detected in HSCs of clinical HSC transplant patients and elderly individuals. A forced increase of intracellular levels of ROS by treatment with a glutathione synthetase inhibitor aggravates the extent of DNA damage, resulting in the functional impairment of HSCs in vivo. The oxidative DNA damage activates the expression of cell-cycle inhibitors in a HSC specific manner, leading to the premature senescence among HSCs, and ultimately to the loss of stem cell function. Importantly, treatment with an antioxidant can antagonize the oxidative DNA damage and eventual HSC dysfunction. The study reveals that ROS play a causative role for DNA damage and the regulation of ROS have a major influence on human HSC aging.

Description: Ex vivo expansion of hematopoietic stem cells (HSC) is a major challenge for clinical and experimental transplantation protocols. However, no significant clinical benefit has been demonstrated to date. Clonal kinetics of ex vivo-expanded HSCs is one of the basic transplantation biology questions to be addressed before we can optimize ex vivo expansion approaches. To characterize human HSC, xenotransplantation techniques such as the severe combined immunodeficiency (SCID) mouse repopulating cell (SRC) assay have proven the most reliable methods thus far. While SRC quantification by limiting dilution analysis (LDA) is the gold standard for measuring in vitro expansion of human HSC, LDA is a statistical method and does not directly establish that a single HSC has self renewed in vitro. By using lentiviral gene-marking and direct intra-bone marrow injection of cultured CD34+ CB cells, we demonstrate here the first direct evidence for self-renewal of individual SRC clones in vitro. To detect multiplied clones, 5x104 gene-marked CD34+ cells were cultured for 4 days in our ex vivo expansion culture system (Exp Hematol, 27:904–915, 1999), and then divided into 10 lots, each of which was transplanted directly into the bone marrow of a NOD/SCID mouse. We used linear amplification-mediated (LAM)-PCR to detect unique genomic-proviral junctions as clonal markers. Detection of the same clones in different mice would provide direct evidence of ex vivo multiplication of a SRC clone. We identified 20 clone-specific genomic-proviral junction sequences by LAM-PCR on 10 mice. Although 14 clones were detected in only one mouse, six clones were detected in more than 2 mice. In the next experiment, purified CD19+EGFP+ and CD33+EGFP+ cells from each mouse were analyzed for each clone to detect multi-lineage differentiation of amplified SRCs. We identified 15 clonal markers from 6 mice. While 12 clones were present in only one mouse, 3 clones were present in 2 independent mice and reconstituted both CD19+and CD33+cells. Finally, we designed a secondary transplantation experiment to confirm the self-renewal ability of each clone. We identified 39 clonal markers from 10 primary and 10 secondary transplanted mice, 11 of which were detected in multiple mice with secondary transplantable ability. Together, of 74 clones analyzed, 20 clones (27%) divided and repopulated in more than two mice after serum-free and stroma-dependent culture. Some of them were secondary transplantable. Furthermore, we identified new class of stem cells based not on repopulation, or cell surface markers, but on response to cytokine stimulation in vitro. Our data demonstrate that current ex vivo expansion conditions result in reliable stem cell expansion and the clonal tracking we have employed is the only reliable method that can be used in the development of clinically appropriate expansion methods.