Utilizing multipotent and self-renewing capabilities, hematopoietic stem cells (HSCs) can maintain hematopoiesis throughout life. However, the mechanism behind such remarkable abilities remains ...undiscovered, at least in part because of the paucity of HSCs and the modest ex vivo expansion of HSCs in media that contain poorly defined albumin supplements such as bovine serum albumin. Here, we describe a simple platform for the expansion of functional mouse HSCs ex vivo for >1 month under fully defined albumin-free conditions. The culture system affords 236- to 899-fold expansion over the course of a month and is also amenable to clonal analysis of HSC heterogeneity. The large numbers of expanded HSCs enable HSC transplantation into nonconditioned recipients, which is otherwise not routinely feasible because of the large numbers of HSCs required. This protocol therefore provides a powerful approach with which to interrogate HSC self-renewal and lineage commitment and, more broadly, to study and characterize the hematopoietic and immune systems.
Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation
, which is a curative therapy for numerous diseases including immunodeficiencies and ...leukaemias
. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche
, stable ex vivo HSC expansion has previously been unattainable
. 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 cultures
; 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.
A specialized bone marrow microenvironment (niche) regulates hematopoietic stem cell (HSC) self-renewal and commitment. For successful donor-HSC engraftment, the niche must be emptied via ...myeloablative irradiation or chemotherapy. However, myeloablation can cause severe complications and even mortality. Here we report that the essential amino acid valine is indispensable for the proliferation and maintenance of HSCs. Both mouse and human HSCs failed to proliferate when cultured in valine-depleted conditions. In mice fed a valine-restricted diet, HSC frequency fell dramatically within 1 week. Furthermore, dietary valine restriction emptied the mouse bone marrow niche and afforded donor-HSC engraftment without chemoirradiative myeloablation. These findings indicate a critical role for valine in HSC maintenance and suggest that dietary valine restriction may reduce iatrogenic complications in HSC transplantation.
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.
DNA replication is spatially and temporally regulated during S‐phase. DNA replication timing is established in early‐G1‐phase at a point referred to as timing decision point. However, how the ...genome‐wide replication timing domains are established is unknown. Here, we show that Rif1 (Rap1‐interacting‐factor‐1), originally identified as a telomere‐binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Depletion of Rif1 results in specific loss of mid‐S replication foci profiles, stimulation of initiation events in early‐S‐phase and changes in long‐range replication timing domain structures. Analyses of replication timing show replication of sequences normally replicating early is delayed, whereas that normally replicating late is advanced, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear‐insoluble structures at late‐M‐to‐early‐G1 and regulates chromatin‐loop sizes. Furthermore, Rif1 colocalizes specifically with the mid‐S replication foci. Thus, Rif1 establishes the mid‐S replication domains that are restrained from being activated at early‐S‐phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher‐order chromatin architecture.
The homologue of the yeast telomeric protein Rif1 regulates the complex temporal programme of human genome replication, possibly controlling chromatin domain establishment at the G1 ‘timing decision point’
Haematopoietic stem cells (HSCs) are arguably the most extensively characterized tissue stem cells. Since the identification of HSCs by prospective isolation, complex multi-parameter flow cytometric ...isolation of phenotypic subsets has facilitated studies on many aspects of HSC biology, including self-renewal, differentiation, ageing, niche, and diversity. Here we demonstrate by unbiased multi-step screening, identification of a single gene, homeobox B5 (Hoxb5, also known as Hox-2.1), with expression in the bone marrow that is limited to long-term (LT)-HSCs in mice. Using a mouse single-colour tri-mCherry reporter driven by endogenous Hoxb5 regulation, we show that only the Hoxb5(+) HSCs exhibit long-term reconstitution capacity after transplantation in primary transplant recipients and, notably, in secondary recipients. Only 7-35% of various previously defined immunophenotypic HSCs are LT-HSCs. Finally, by in situ imaging of mouse bone marrow, we show that >94% of LT-HSCs (Hoxb5(+)) are directly attached to VE-cadherin(+) cells, implicating the perivascular space as a near-homogenous location of LT-HSCs.
Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of ...haematological diseases
. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation
. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo
. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.
In vitro generation of hematopoietic stem cells (HSCs) from induced pluripotent stem cells (iPSCs) has the potential to provide novel therapeutic approaches for replacing bone marrow (BM) ...transplantation without rejection or graft versus host disease. Hitherto, however, it has proved difficult to generate truly functional HSCs transplantable to adult host mice. Here, we demonstrate a unique in vivo differentiation system yielding engraftable HSCs from mouse and human iPSCs in teratoma-bearing animals in combination with a maneuver to facilitate hematopoiesis. In mice, we found that iPSC-derived HSCs migrate from teratomas into the BM and their intravenous injection into irradiated recipients resulted in multilineage and long-term reconstitution of the hematolymphopoietic system in serial transfers. Using this in vivo generation system, we could demonstrate that X-linked severe combined immunodeficiency (X-SCID) mice can be treated by HSCs derived from gene-corrected clonal iPSCs. It should also be noted that neither leukemia nor tumors were observed in recipients after transplantation of iPSC-derived HSCs. Taken our findings together, our system presented in this report should provide a useful tool not only for the study of HSCs, but also for practical application of iPSCs in the treatment of hematologic and immunologic diseases.
Hematopoietic stem cells (HSCs) exhibit considerable cell-intrinsic changes with age. Here, we present an integrated analysis of transcriptome and chromatin accessibility of aged HSCs and downstream ...progenitors. Alterations in chromatin accessibility preferentially take place in HSCs with aging, which gradually resolve with differentiation. Differentially open accessible regions (open DARs) in aged HSCs are enriched for enhancers and show enrichment of binding motifs of the STAT, ATF, and CNC family transcription factors that are activated in response to external stresses. Genes linked to open DARs show significantly higher levels of basal expression and their expression reaches significantly higher peaks after cytokine stimulation in aged HSCs than in young HSCs, suggesting that open DARs contribute to augmented transcriptional responses under stress conditions. However, a short-term stress challenge that mimics infection is not sufficient to induce persistent chromatin accessibility changes in young HSCs. These results indicate that the ongoing and/or history of exposure to external stresses may be epigenetically inscribed in HSCs to augment their responses to external stimuli.
The hard-to-culture slightly halophilic myxobacterium "
" SMH-27-4 produces antifungal cyclodepsipeptide miuraenamide A (
). Herein, the region (85.9 kbp) containing the biosynthetic gene cluster ...(BGC) coding the assembly of
was identified and heterologously expressed in
A biosynthetic pathway proposed using in silico analysis was verified through the gene disruption of the heterologous transformant. In addition to the core polyketide synthase (PKS) and nonribosomal peptide synthase (NRPS) genes, tyrosine halogenase and
-methyltransferase genes participated in the biosynthesis of
as their gene-disrupted mutants produced a new congener, debromomiuraenamide A (
), and a previously isolated congener, miuraenamide E (
), respectively. Multigene disruption provided a heterologous mutant that produced
with the highest yield among the prepared mutants. When fed on 3-bromo-L-tyrosine, this mutant produced more
in the yield of 1.21 mg/L, which was 20 times higher than that produced by the initially prepared heterologous transformant. Although this yield was comparable to that of the original producer SMH-27-4 (1 mg/L), the culture time was 4.5 times shorter than that of SMH-27-4, indicating a five-fold efficiency in productivity. The results indicate the great potential of the miuraenamide BGC for the future contribution to drug development through logical gene manipulation.
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