Non-conditioned bone marrow chimeric mouse generation using culture-based enrichment of hematopoietic stem and progenitor cells.

Bone marrow (BM) chimeric mice are a valuable tool in the field of immunology, with the genetic manipulation of donor cells widely used to study gene function under physiological and pathological settings. To date, however, BM chimera protocols require myeloablative conditioning of recipient mice, which dramatically alters steady-state hematopoiesis. Additionally, most protocols use fluorescence-activated cell sorting (FACS) of hematopoietic stem/progenitor cells (HSPCs) for ex vivo genetic manipulation. Here, we describe our development of cell culture techniques for the enrichment of functional HSPCs from mouse BM without the use of FACS purification. Furthermore, the large number of HSPCs derived from these cultures generate BM chimeric mice without irradiation. These HSPC cultures can also be genetically manipulated by viral transduction, to allow for doxycycline-inducible transgene expression in donor-derived immune cells within non-conditioned immunocompetent recipients. This technique is therefore expected to overcome current limitations in mouse transplantation models.

Author Correction: Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation.

Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation1, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias2. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche3-5, stable ex vivo HSC expansion has previously been unattainable6,7. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures8; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.

Interleukin-12 supports in vitro self-renewal of long-term hematopoietic stem cells.

Hematopoietic stem cells (HSCs) self-renew or differentiate through division. Cytokines are essential for inducing HSC division, but the optimal cytokine combination to control self-renewal of HSC in vitro remains unclear. In this study, we compared the effects of interleukin-12 (IL-12) and thrombopoietin (TPO) in combination with stem cell factor (SCF) on in vitro self-renewal of HSCs. Single-cell assays were used to overcome the heterogeneity issue of HSCs, and serum-free conditions were newly established to permit reproduction of data. In single-cell cultures, CD150+CD48-CD41-CD34-c-Kit+Sca-1+lineage- HSCs divided significantly more slowly in the presence of SCF+IL-12 compared with cells in the presence of SCF+TPO. Serial transplantation of cells from bulk and clonal cultures revealed that TPO was more effective than IL-12 at supporting in vitro self-renewal of short-term (<6 months) HSCs, resulting in a monophasic reconstitution wave formation, whereas IL-12 was more effective than TPO at supporting the in vitro self-renewal of long-term (>6 months) HSCs, resulting in a biphasic reconstitution wave formation. The control of division rate in HSCs appeared to be crucial for preventing the loss of self-renewal potential from their in vitro culture.

Fine-scale geographic clustering pattern of human T-cell leukemia virus type 1 infection among blood donors in Kyushu-Okinawa, Japan.

Human T-cell leukemia virus type I (HTLV-1) infection is endemic in Japan, particularly clustered in the southwestern district, Kyushu-Okinawa, which consists of eight prefectures that further consist of 274 municipalities. However, no information is available about the fine-scale distribution of HTLV-1 infection within Kyushu-Okinawa. To assess the municipal-level distribution of people with HTLV-1 infection in Kyushu-Okinawa, we performed a cross-sectional study using a fine-scale geographic information system map based on HTLV-1 screening test results from the Japanese Red Cross database from September 2012 to February 2014. Of the 881 871 (646 914 male, 234 957 female) screened blood donors, 981 were seropositive for HTLV-1 by confirmatory test. The seroprevalence was 0.11% (95% confidence interval [CI] 0.10%-0.12%) for all, 0.094% (95% CI, 0.09%-0.10%) for male, and 0.16% (95% CI, 0.14%-0.18%) for female individuals. The sex- and age-specific HTLV-1 seroprevalence varied significantly across municipalities; particularly, the seroprevalence among women aged 50 years was significantly higher than that of men in both the mainland of Kyushu-Okinawa and the satellite island, in all of which the seroprevalence of HTLV-1 was more than 1.2%. These results show that, even in the Kyushu-Okinawa district, there are endemic clusters of HTLV-1 in small areas. This suggests that public health education programs are needed to eliminate new HTLV-1 infection in these areas.

Branched-chain amino acid depletion conditions bone marrow for hematopoietic stem cell transplantation avoiding amino acid imbalance-associated toxicity.

Hematopoietic stem cells (HSCs) are used clinically in bone marrow (BM) transplantation due to their unique ability to reform the entire hematopoietic system. Recently, we reported that HSCs are highly sensitive to valine, one of the three branched-chain amino acids (BCAAs) in addition to isoleucine and leucine. Dietary depletion of valine could even be used as a conditioning regimen for HSC transplantation. Here, we report that HSCs are highly sensitive to the balance of BCAAs, with both proliferation and survival reduced by BCAA imbalance. However, low but balanced BCAA levels failed to rescue HSC maintenance. Importantly, in vivo depletion of all three BCAAs was significantly less toxic than depletion of valine only. We demonstrate that BCAA depletion can replace valine depletion as a safer alternative to BM conditioning. In summary, by determining HSC metabolic requirements, we can improve metabolic approaches to BM conditioning.

Physiological Srsf2 P95H expression causes impaired hematopoietic stem cell functions and aberrant RNA splicing in mice.

Splicing factor mutations are characteristic of myelodysplastic syndromes (MDS) and related myeloid neoplasms and implicated in their pathogenesis, but their roles in the development of MDS have not been fully elucidated. In the present study, we investigated the consequence of mutant Srsf2 expression using newly generated Vav1-Cre-mediated conditional knockin mice. Mice carrying a heterozygous Srsf2 P95H mutation showed significantly reduced numbers of hematopoietic stem and progenitor cells (HSPCs) and differentiation defects both in the steady-state condition and transplantation settings. Srsf2-mutated hematopoietic stem cells (HSCs) showed impaired long-term reconstitution compared with control mice in competitive repopulation assays. Although the Srsf2 mutant mice did not develop MDS under the steady-state condition, when their stem cells were transplanted into lethally irradiated mice, the recipients developed anemia, leukopenia, and erythroid dysplasia, which suggests the role of replicative stress in the development of an MDS-like phenotype in Srsf2-mutated mice. RNA sequencing of the Srsf2-mutated HSPCs revealed a number of abnormal splicing events and differentially expressed genes, including several potential targets implicated in the pathogenesis of hematopoietic malignancies, such as Csf3r, Fyn, Gnas, Nsd1, Hnrnpa2b1, and Trp53bp1 Among the mutant Srsf2-associated splicing events, most commonly observed were the enhanced inclusion and/or exclusion of cassette exons, which were caused by the altered consensus motifs for the recognition of exonic splicing enhancers. Our findings suggest that the mutant Srsf2 leads to a compromised HSC function by causing abnormal RNA splicing and expression, contributing to the deregulated hematopoiesis that recapitulates the MDS phenotypes, possibly as a result of additional genetic and/or environmental insults.

Analysis of HTLV-1 proviral load (PVL) and antibody detected with various kinds of tests in Japanese blood donors to understand the relationship between PVL and antibody level and to gain insights toward better antibody testing.

Adult T-cell leukemia/lymphoma (ATL) occurs in approximately 5% of individuals infected with human T-cell leukemia virus type 1 (HTLV-1). A high proviral load (PVL; more than four copies per 100 peripheral blood mononuclear cells (PBMCs) or 1.6 copies per 100 blood leukocytes) and being male are risk factors for ATL development. Whether anti-HTLV-1 antibody level is related to such risk is unknown. Here, PVL and antibody levels were examined using real-time PCR and other tests in 600 HTLV-1 positive screened Japanese blood donors to understand the relationship between PVL and antibody level in asymptomatic carriers and to gain insights toward better antibody testing for HTLV-1 infection. The 430 donors in whom proviral DNA was detected were considered as true positives for HTLV-1 infection. Among donors aged 40 years or older, more males than females had a PVL corresponding to more than 1.6% infected leukocytes, and an antibody titer below the median (P = 0.0018). In antibody tests using an HTLV-1 positive cell line or Env antigens there was a large discrepancy in antibody titer among 13 provirus-positive samples, probably suggesting that antibody-based screening tests should incorporate multiple HTLV-1 antigens, such as Gag and Env antigens.

An All-Recombinant Protein-Based Culture System Specifically Identifies Hematopoietic Stem Cell Maintenance Factors.

Hematopoietic stem cells (HSCs) are considered one of the most promising therapeutic targets for the treatment of various blood disorders. However, due to difficulties in establishing stable maintenance and expansion of HSCs in vitro, their insufficient supply is a major constraint to transplantation studies. To solve these problems we have developed a fully defined, all-recombinant protein-based culture system. Through this system, we have identified hemopexin (HPX) and interleukin-1α as responsible for HSC maintenance in vitro. Subsequent molecular analysis revealed that HPX reduces intracellular reactive oxygen species levels within cultured HSCs. Furthermore, bone marrow immunostaining and 3D immunohistochemistry revealed that HPX is expressed in non-myelinating Schwann cells, known HSC niche constituents. These results highlight the utility of this fully defined all-recombinant protein-based culture system for reproducible in vitro HSC culture and its potential to contribute to the identification of factors responsible for in vitro maintenance, expansion, and differentiation of stem cell populations.