技术资料

Elucidation of mechanisms that regulate hematopoietic stem cell self-renewal and differentiation would be facilitated by the identification of defined culture conditions that allow these cells to be amplified. We now demonstrate a significant net increase (3-fold,P textless 0.001) in vitro of cells that are individually able to permanently and competitively reconstitute the lymphoid and myeloid systems of syngeneic recipient mice when Sca-1(+)lin- adult marrow cells are incubated for 10 days in serum-free medium with interleukin 11,flt3-ligand,and Steel factor. Moreover,the culture-derived repopulating cells continued to expand their numbers in the primary hosts at the same rate seen in recipients of noncultured stem cells. In the expansion cultures,long-term culture-initiating cells increased 7- +/- 2-fold,myeloid colony-forming cells increased 140- +/- 36-fold,and total nucleated cells increased 230- +/- 62-fold. Twenty-seven of 100 cultures initiated with 15 Sca-1(+)lin- marrow cells were found to contain transplantable stem cells 10 days later. This frequency of positive cultures is the same as the frequency of transplantable stem cells in the original input suspension,suggesting that most had undergone at least one self-renewal division in vitro. No expansion of stem cells was seen when Sca-1+TER119- CD34+ day 14.5 fetal liver cells were cultured under the same conditions. These findings set the stage for further investigations of the mechanisms by which cytokine stimulation may elicit different outcomes in mitotically activated hematopoietic stem cells during ontogeny and in the adult. View Publication

A large number of biologic,technological,and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long-term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures,and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established,long-term marrow cultures of female origin. At 4 weeks,cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly,for 3 of 4 long-term cultures,multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long-term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore,such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion. View Publication

Human hematopoiesis originates in a population of stem cells with transplantable lympho-myeloid reconstituting potential,but a method for quantitating such cells has not been available. We now describe a simple assay that meets this need. It is based on the ability of sublethally irradiated immunodeficient nonobese diabetic-scid/scid (NOD/SCID) mice to be engrafted by intravenously injected human hematopoietic cells and uses limiting dilution analysis to measure the frequency of human cells that produce both CD34(-)CD19(+) (B-lymphoid) and CD34(+) (myeloid) colony-forming cell progeny in the marrow of such recipients 6 to 8 weeks post-transplant. Human cord blood (CB) contains approximately 5 of these competitive repopulating units (CRU) per ml that have a similar distribution between the CD38(-) and CD38(+) subsets of CD34(+) CB cells as long-term culture-initiating cells (LTC-IC) (4:1 vs. 2:1). Incubation of purified CD34(+)CD38(-) human CB cells in serum-free medium containing flt-3 ligand,Steel factor,interleukin 3,interleukin 6,and granulocyte colony-stimulating factor for 5-8 days resulted in a 100-fold expansion of colony-forming cells,a 4-fold expansion of LTC-IC,and a 2-fold (but significant,P textless 0.02) increase in CRU. The culture-derived CRU,like the original CB CRU,generated pluripotent,erythroid,granulopoietic,megakaryopoietic,and pre-B cell progeny upon transplantation into NOD/SCID mice. These findings demonstrate an equivalent phenotypic heterogeneity amongst human CB cells detectable as CRU and LTC-IC. In addition,their similarly modest response to stimulation by a combination of cytokines that extensively amplify LTC-IC from normal adult marrow underscores the importance of ontogeny-dependent changes in human hematopoietic stem cell proliferation and self-renewal. View Publication

Juvenile dermatomyositis (JDM) is a pediatric autoimmune disease associated with characteristic rash and proximal muscle weakness. To gain insight into differential lymphocyte gene expression in JDM,peripheral blood mononuclear cells from 4 new-onset JDM patients and 4 healthy controls were sorted into highly enriched lymphocyte populations for RNAseq analysis. NK cells from JDM patients had substantially greater differentially expressed genes (273) than T (57) and B (33) cells. Upregulated genes were associated with the innate immune response and cell cycle,while downregulated genes were associated with decreased ribosomal RNA. Suppressed ribosomal RNA in JDM NK cells was validated by measuring transcription and phosphorylation levels. We confirmed a population of low ribosome expressing NK cells in healthy adults and children. This population of low ribosome NK cells was substantially expanded in 6 treatment-na{\{i}}ve JDM patients and was associated with decreased NK cell degranulation. The enrichment of this NK low ribosome population was completely abrogated in JDM patients with quiescent disease. Together these data suggest NK cells are highly activated in new-onset JDM patients with an increased population of low ribosome expressing NK cells which correlates with decreased NK cell function and resolved with control of active disease." View Publication

We have investigated the potential of stirred suspension cultures to support hematopoiesis from starting innocula of normal human bone marrow cells. Initial studies showed that the short-term maintenance of both colony-forming cell (CFC) numbers and their precursors,detected as long-term culture-initiating cells (LTC-IC),could be achieved as well in stirred suspension cultures as in static cultures. Neither of these progenitor cell populations was affected in either type of culture when porous microcarriers were added to provide an increased surface for adherent cell attachment. Supplementation of the medium with 10 ng/ml of Steel factor (SF) and 2 ng/ml of interleukin-3 (IL-3) resulted in a significant expansion of LTC-IC,CFC and total cell numbers in stirred cultures. Both the duration and ultimate magnitude of these expansions were correlated with the initial cell density and after 4 weeks the number of LTC-IC and CFC present in stirred cultures initiated with the highest starting cell concentration tested reflected average increases of 7- and 22-fold,respectively,above input values. Stirred suspension cultures offer the combined advantages of homogeneity and lack of dependence on the formation and maintenance of an adherent cell layer. Our results suggest their applicability to the development of scaled-up bioreactor systems for clinical procedures requiring the production of primitive hematopoietic cell populations. In addition,stirred suspension cultures may offer a new tool for the analysis of hematopoietic regulatory mechanisms. View Publication

It has been proposed that bone marrow (BM) hematopoietic stem and progenitor cells are distributed along an oxygen (O2) gradient,where stem cells reside in the most hypoxic areas and proliferating progenitors are found in O2-rich areas. However,the effects of hypoxia on human hematopoietic stem cells (HSCs) have not been characterized. Our objective was to evaluate the functional and molecular responses of human BM progenitors and stem cells to hypoxic conditions. BM lineage-negative (Lin-) CD34+CD38- cells were cultured in serum-free medium under 1.5% O2 (hypoxia) or 20% O2 (normoxia) for 4 days. Using limiting dilution analysis,we demonstrate that the absolute number of SCID-repopulating cells (SRCs) increased by 5.8-fold in hypoxic cultures compared with normoxia,and by 4.2-fold compared with freshly isolated Lin-CD34+CD38- cells. The observed increase in BM-repopulating activity was associated with a preferential expansion of Lin-CD34+CD38- cells. We also demonstrate that,in response to hypoxia,hypoxia-inducible factor-1alpha protein was stabilized,surface expression of angiogenic receptors was upregulated,and VEGF secretion increased in BM Lin-CD34+ cultures. The use of low O2 levels to enhance the survival and/or self-renewal of human BM HSCs in vitro represents an important advance and could have valuable clinical implications. View Publication

The contribution of B cells to the pathology of Omenn syndrome and leaky severe combined immunodeficiency (SCID) has not been previously investigated. We have studied a mut/mut mouse model of leaky SCID with a homozygous Rag1 S723C mutation that impairs,but does not abrogate,V(D)J recombination activity. In spite of a severe block at the pro-B cell stage and profound B cell lymphopenia,significant serum levels of immunoglobulin (Ig) G,IgM,IgA,and IgE and a high proportion of Ig-secreting cells were detected in mut/mut mice. Antibody responses to trinitrophenyl (TNP)-Ficoll and production of high-affinity antibodies to TNP-keyhole limpet hemocyanin were severely impaired,even after adoptive transfer of wild-type CD4(+) T cells. Mut/mut mice produced high amounts of low-affinity self-reactive antibodies and showed significant lymphocytic infiltrates in peripheral tissues. Autoantibody production was associated with impaired receptor editing and increased serum B cell-activating factor (BAFF) concentrations. Autoantibodies and elevated BAFF levels were also identified in patients with Omenn syndrome and leaky SCID as a result of hypomorphic RAG mutations. These data indicate that the stochastic generation of an autoreactive B cell repertoire,which is associated with defects in central and peripheral checkpoints of B cell tolerance,is an important,previously unrecognized,aspect of immunodeficiencies associated with hypomorphic RAG mutations. View Publication

To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies,efficient and scalable expansion procedures must be developed. For other adherent human cell types,the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study,a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings,indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( N s 1 u ),thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics,computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner,improved process understanding allowed an expansion factor of ≈ 26 to be achieved,yielding more than 3 × 10 9 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability,identity,and differentiation potential throughout cultivation. • N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs The online version contains supplementary material available at 10.1007/s00253-024-13372-3. View Publication

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However,achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L -scale,others,notably instrumented SU multiplate and fixed-bed bioreactors,remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods,operating ranges were identified for the Xpansion ® 10 and Ascent™ 1 m 2 bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application,almost 5 × 10 9 viable cells could be produced within 5 days,achieving expansion factors of up to 35 without discernable impact on cell viability,identity,or differentiation potential. Key Points • Bioengineering characterizations allowed the identification of operating ranges that supported satisfactory hiPSC expansion • Both the Xpansion ® 10 multiplate and Ascent™ 1 m 2 fixed-bed reactor accommodated the production of almost 5 × 10 9 viable cells within 5 days • Exposing the hiPSCs to a median wall shear stress of up to 8.2 × 10 −5 N cm −2 did not impair quality The online version contains supplementary material available at 10.1007/s00253-024-13373-2. View Publication

Oligodendrocytes-the myelin-forming cells of the central nervous system-can be regenerated during adulthood. In adults,new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs),but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized,the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1,a protein deacetylase implicated in energy metabolism,increases the production of new OPCs in the adult mouse brain,in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally,generating fully myelinating oligodendrocytes. Remarkably,SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling,in particular PDGF receptor α (PDGFRα). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK-signalling molecules downstream of PDGFRα. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases,such as multiple sclerosis. View Publication

OBJECTIVE: The T-cell receptor zeta (TCR zeta)-chain is a master sensor and regulator of lymphocyte responses. Loss of TCR zeta-chain expression has been documented during infectious and inflammatory diseases and defines a population of effector T cells (TCR zeta(dim) T cells) that migrate to inflamed tissues. We assessed the expression and functional correlates of circulating TCR zeta(dim) T cells in coronary artery disease. METHODS AND RESULTS: We examined the expression of TCR zeta-chain by flow cytometry in 140 subjects. Increased peripheral blood CD4(+) TCR zeta(dim) T cells were found in patients with acute coronary syndromes (ACS,n=66; median 5.3%,interquartile 2.6 to 9.1% of total CD4(+) T cells; Ptextless0.0001) compared to chronic stable angina (CSA,n=32; 1.6%; 1.0 to 4.1%) and controls (n=42; 1.5%; 0.5 to 2.9%). Such increase was significantly greater in ACS patients with elevated levels of C-reactive protein,and it persisted after the acute event. Moreover,TCR zeta(dim) cells were also more represented within CD8(+) T cell,NK,and CD4(+)CD28(null) T cell subsets in ACS compared to CSA and controls. Finally,CD4(+) and CD8(+) TCR zeta(dim) T cells isolated from ACS displayed an enhanced transendothelial migratory capacity. CONCLUSIONS: TCR zeta(dim) T cells,an effector T-cell subset with transendothelial migratory ability,are increased in ACS,and may be implicated in coronary instability. View Publication

The repurposed CRISPR-Cas9 system has recently emerged as a revolutionary genome-editing tool. Here we report a modification in the expression of the guide RNA (gRNA) required for targeting that greatly expands the targetable genome. gRNA expression through the commonly used U6 promoter requires a guanosine nucleotide to initiate transcription,thus constraining genomic-targeting sites to GN19NGG. We demonstrate the ability to modify endogenous genes using H1 promoter-expressed gRNAs,which can be used to target both AN19NGG and GN19NGG genomic sites. AN19NGG sites occur ˜15% more frequently than GN19NGG sites in the human genome and the increase in targeting space is also enriched at human genes and disease loci. Together,our results enhance the versatility of the CRISPR technology by more than doubling the number of targetable sites within the human genome and other eukaryotic species. View Publication