Reduced repression of cytokine signaling ameliorates age‐induced decline in hematopoietic stem cell function

Aging causes profound effects on the hematopoietic stem cell (HSC) pool, including an altered output of mature progeny and enhanced self‐propagation of repopulating‐defective HSCs. An important outstanding question is whether HSCs can be protected from aging. The signal adaptor protein LNK negatively regulates hematopoiesis at several cellular stages. It has remained unclear how the enhanced sensitivity to cytokine signaling caused by LNK deficiency affects hematopoiesis upon aging. Our findings demonstrate that aged LNK^?/? HSCs displayed a robust overall reconstitution potential and gave rise to a hematopoietic system with a balanced lineage distribution. Although aged LNK^?/? HSCs displayed a distinct molecular profile in which reduced proliferation was central, little or no difference in the proliferation of aged LNK^?/? HSCs was observed after transplantation when compared to aged WT HSCs. This coincided with equal telomere maintenance in WT and LNK^?/? HSCs. Collectively, our studies suggest that enhanced cytokine signaling can counteract functional age‐related HSC decline.     

Gene profiling reveals association between altered Wnt signaling and loss of T‐cell potential with age in human hematopoietic stem cells

Functional decline of the hematopoietic system occurs during aging and contributes to clinical consequences, including reduced competence of adaptive immunity and increased incidence of myeloid diseases. This has been linked to aging of the hematopoietic stem cell (HSC) compartment and has implications for clinical hematopoietic cell transplantation as prolonged periods of T‐cell deficiency follow transplantation of adult mobilized peripheral blood (PB), the primary transplant source. Here, we examined the gene expression profiles of young and aged HSCs from human cord blood and adult mobilized PB, respectively, and found that Wnt signaling genes are differentially expressed between young and aged human HSCs, with less activation of Wnt signaling in aged HSCs. Utilizing the OP9‐DL1 in vitro co‐culture system to promote T‐cell development under stable Notch signaling conditions, we found that Wnt signaling activity is important for T‐lineage differentiation. Examination of Wnt signaling components and target gene activation in young and aged human HSCs during T‐lineage differentiation revealed an association between reduced Wnt signal transduction, increasing age, and impaired or delayed T‐cell differentiation. This defect in Wnt signal activation of aged HSCs appeared to occur in the early T‐progenitor cell subset derived during in vitro T‐lineage differentiation. Our results reveal that reduced Wnt signaling activity may play a role in the age‐related intrinsic defects of aged HSCs and early hematopoietic progenitors and suggest that manipulation of this pathway could contribute to the end goal of improving T‐cell generation and immune reconstitution following clinical transplantation.     

Angiotensin II modulates the murine hematopoietic stem cell and progenitors cocultured with stromal S17 cells

Although the existence of the renin–angiotensin system (RAS) in the bone marrow is clear, the exact role of this system in hematopoiesis has not yet been fully characterized. Here the direct role of angiotensin II (AngII) in hematopoietic stem cells (HSCs), common myeloid progenitors (CMPs), granulocyte/monocyte progenitors (GMPs), and megakaryocytes/erythroid progenitors (MEPs), using a system of coculture with stromal S17 cells. Flow cytometry analysis showed that AngII increases the percentage of HSC and GMP, while reducing CMP with no effect on MEP. According to these data, AngII increased the total number of mature Gr-1⁺/Mac-1⁺ cells without changes in Terr119⁺ cells. AngII does not induce cell death in the population of LSK cells. In these populations, treatment with AngII decreases the expression of Ki67⁺ protein with no changes in the Notch1 expression, suggesting a role for AngII on the quiescence of immature cells. In addition, exposure to AngII from murine bone marrow cells increased the number of CFU-GM and BFU-E in a clonogenic assay. In conclusion, our data showed that AngII is involved in the regulation of hematopoiesis with a special role in HSC, suggesting that AngII should be evaluated in coculture systems, especially in cases that require the expansion of these cells in vitro, still a significant challenge for therapeutic applications in humans.     

Smurf2 regulates hematopoietic stem cell self‐renewal and aging

The age‐dependent decline in the self‐renewal capacity of stem cells plays a critical role in aging, but the precise mechanisms underlying this decline are not well understood. By limiting proliferative capacity, senescence is thought to play an important role in age‐dependent decline of stem cell self‐renewal, although direct evidence supporting this hypothesis is largely lacking. We have previously identified the E3 ubiquitin ligase Smurf2 as a critical regulator of senescence. In this study, we found that mice deficient in Smurf2 had an expanded hematopoietic stem cell (HSC) compartment in bone marrow under normal homeostatic conditions, and this expansion was associated with enhanced proliferation and reduced quiescence of HSCs. Surprisingly, increased cycling and reduced quiescence of HSCs in Smurf2‐deficient mice did not lead to premature exhaustion of stem cells. Instead, HSCs in aged Smurf2‐deficient mice had a significantly better repopulating capacity than aged wild‐type HSCs, suggesting that decline in HSC function with age is Smurf2 dependent. Furthermore, Smurf2‐deficient HSCs exhibited elevated long‐term self‐renewal capacity and diminished exhaustion in serial transplantation. As we found that the expression of Smurf2 was increased with age and in response to regenerative stress during serial transplantation, our findings suggest that Smurf2 plays an important role in regulating HSC self‐renewal and aging.     

In senescence,age‐associated B cells secrete TNFα and inhibit survival of B‐cell precursors*

Aged mice exhibit ~ 5–10‐fold increases in an ordinarily minor CD21/35^? CD23^? mature B‐cell subset termed age‐associated B cells (ABCs). ABCs from old, but not young, mice induce apoptosis in pro‐B cells directly through secretion of TNFα. In addition, aged ABCs, via TNFα, stimulate bone marrow cells to suppress pro‐B‐cell growth. ABC effects can be prevented by the anti‐inflammatory cytokine IL‐10. Notably, CD21/35⁺ CD23⁺ follicular (FO) splenic and FO‐like recirculating bone marrow B cells in both young and aged mice contain a subpopulation that produces IL‐10. Unlike young adult FO B cells, old FO B cells also produce TNFα; however, secretion of IL‐10 within this B‐cell population ameliorates the TNFα‐mediated effects on B‐cell precursors. Loss of B‐cell precursors in the bone marrow of old mice in vivo was significantly associated with increased ABC relative to recirculating FO‐like B cells. Adoptive transfer of aged ABC into RAG‐2 KO recipients resulted in significant losses of pro‐B cells within the bone marrow. These results suggest that alterations in B‐cell composition during old age, in particular, the increase in ABC within the B‐cell compartments, contribute to a pro‐inflammatory environment within the bone marrow. This provides a mechanism of inappropriate B‐cell ‘feedback’ that promotes down‐regulation of B lymphopoiesis in old age.     

Temporal multimodal single-cell profiling of native hematopoiesis illuminates altered differentiation trajectories with age

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Compromised steady‐state germinal center activity with age in nonhuman primates

Age‐related reductions in vaccine‐induced B cells in aging indicate that germinal centers (GCs), the anatomical site where the development of humoral responses takes place, may lose efficacy with age. We have investigated the baseline follicular and GC composition in nonhuman primates (NHPs) with respect to their age. There was a marked reduction in follicular area in old animals. We found significantly lower normalized numbers of follicular PD1^hi CD4 T (Tfh) and proliferating (Ki67^hi) GC B cells with aging, a profile associated with significantly higher numbers of potential follicular suppressor FoxP3^hiLag3^hi CD4 T cells. Furthermore, a positive correlation was found between Tfh and follicular CD8 T cells (fCD8) only in young animals. Despite the increased levels of circulating preinflammatory factors in aging, young animals had higher numbers of monocytes and granulocytes in the follicles, a profile negatively associated with numbers of Tfh cells. Multiple regression analysis showed an altered association between GC B cells and other GC immune cell populations in old animals suggesting a differential mechanistic regulation of GC activity in aging. Our data demonstrate defective baseline GC composition in old NHPs and provide an immunological base for further understanding the adaptive humoral responses with respect to aging.     

In aged mice,low surrogate light chain promotes pro‐B‐cell apoptotic resistance,compromises the PreBCR checkpoint,and favors generation of autoreactive,phosphorylcholine‐specific B cells

In aged mice, new B‐cell development is diminished and the antibody repertoire becomes more autoreactive. Our studies suggest that (i) apoptosis contributes to reduced B lymphopoiesis in old age and preferentially eliminates those B‐cell precursors with higher levels of the surrogate light chain (SLC) proteins (λ5/VpreB) and (ii) λ5^low B‐cell precursors generate new B cells which show increased reactivity to the self‐antigen/bacterial antigen phosphorylcholine (PC). Pro‐B cells in old bone marrow as well as pro‐B cells from young adult λ5‐deficient mice are resistant to cytokine‐induced apoptosis (TNFα; TGFβ), indicating that low λ5 expression in pro‐B cells is sufficient to cause increased survival. Transfer of TNFα‐producing ‘age‐associated B cells’ (ABC; CD21/35^? CD23^?) or follicular (FO) B cells from aged mice into RAG‐2 KO recipients led to preferential loss of λ5^high pro‐B cells, but retention of λ5^low, apoptosis‐resistant pro‐B cells. In old mice, there is increased reactivity to PC in both immature bone marrow B cells and mature splenic FO B cells. In young mice, absence of λ5 expression led to a similar increase in PC reactivity among bone marrow and splenic B cells. We propose that in old age, increased apoptosis, mediated in part by TNFα‐producing B cells, results in preferential loss of SLC^high pro‐B cells within the bone marrow. Further B‐cell development then occurs via an ‘SLC^low’ pathway that not only impairs B‐cell generation, but promotes autoreactivity within the naïve antibody repertoires in the bone marrow and periphery.     

Microbubble‐mediated sonoporation for highly efficient transfection of recalcitrant human B‐ cell lines

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult‐to‐transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B‐cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP‐positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP‐positive cells, respectively. Here, we present sonoporation as a feasible non‐viral strategy for stable and highly efficient heterologous transfection of recalcitrant B‐cell lines. This is the first demonstration of a non‐viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B‐cell lines.     

Fibronectin promotes differentiation of neural crest progenitors endowed with smooth muscle cell potential

The neural crest (NC) is a model system used to investigate multipotency during vertebrate development. Environmental factors control NC cell fate decisions. Despite the well-known influence of extracellular matrix molecules in NC cell migration, the issue of whether they also influence NC cell differentiation has not been addressed at the single cell level. By analyzing mass and clonal cultures of mouse cephalic and quail trunk NC cells, we show for the first time that fibronectin (FN) promotes differentiation into the smooth muscle cell phenotype without affecting differentiation into glia, neurons, and melanocytes. Time course analysis indicated that the FN-induced effect was not related to massive cell death or proliferation of smooth muscle cells. Finally, by comparing clonal cultures of quail trunk NC cells grown on FN and collagen type IV (CLIV), we found that FN strongly increased both NC cell survival and the proportion of unipotent and oligopotent NC progenitors endowed with smooth muscle potential. In contrast, melanocytic progenitors were prominent in clonogenic NC cells grown on CLIV. Taken together, these results show that FN promotes NC cell differentiation along the smooth muscle lineage, and therefore plays an important role in fate decisions of NC progenitor cells.     

Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential

Neural stem (NS) cells are multipotent cells defined by their capacity to proliferate and differentiate into all neuronal and glial phenotypes. NS cells can be obtained from specific regions of the adult brain, or generated from embryonic stem cells (ESCs). NS cells differentiate into neural progenitor (NP) cells and subsequently neural precursors, as transient steps towards terminal differentiation into specific mature neuronal or glial phenotypes. When cultured in EGF and FGF2, ESC-derived NS cells have been reported to be stable and multipotent. Conditions that enable differentiation of NS cells through the committed progenitor and precursor stages to specific neuronal subtypes have not been fully established. In this study we investigated, using Lmx1a reporter ESCs, whether the length of neural induction (NI) dictated the phenotypic potential of cultures of ESC-derived NS cells or NP cells. Following 4, 7 or 10 day periods of NI, ESCs in monolayer culture were harvested and cultured as neurospheres, prior to replating as monolayer cultures for several passages in EGF and FGF2. The NS/NP cultures were then directed towards mature neuronal fates over 16-17 days. 4 and 7-day NS cell cultures could not be differentiated towards dopaminergic, serotonergic or cholinergic fates as determined by the absence of tyrosine hydroxylase, 5-HT or choline acetyltransferase (ChAT) immunolabelling. In contrast NS/NP cultures derived after 10 days of NI were able to generate tyrosine hydroxylase and 5-HT positive neurons (24 ± 6 and 13 ± 1% of the βIII-tubulin positive population, respectively, n = 3). Our data suggest that extended periods of neural induction enhanced the potential of mouse ESC-derived NS/NP cells to generate specific subtypes of neurons. NS/NP cells derived after shorter periods of NI appeared to be lineage-restricted in relation to the neuronal subtypes observed after removal of EGF.     

Ectopic expression of ROR1 prevents cochlear hair cell loss in guinea pigs with noise‐induced hearing loss

Noise‐induced hearing loss (NIHL) is one of the most frequent disabilities in industrialized countries. Evidence shows that hair cell loss in the auditory end organ is responsible for the majority of various ear pathological conditions. The functional roles of the receptor tyrosine kinase ROR1 have been underscored in various tumours. In this study, we evaluated the ability of ROR1 to influence cochlear hair cell loss of guinea pigs with NIHL. The NIHL model was developed in guinea pigs, with subsequent measurement of the auditory brainstem response (ABR). Gain‐of‐function experiments were employed to explore the role of ROR1 in NIHL. The interaction between ROR1 and Wnt5a and their functions in the cochlear hair cell loss were further analysed in response to alteration of ROR1 and Wnt5a. Guinea pigs with NIHL demonstrated elevated ABR threshold and down‐regulated ROR1, Wnt5a and NF‐κB p65. The up‐regulation of ROR1 was shown to decrease the cochlear hair cell loss and the expression of pro‐apoptotic gene (Bax, p53) in guinea pig cochlea, but promoted the expression of anti‐apoptotic gene (Bcl‐2) and the fluorescence intensity of cleaved‐caspase‐3. ROR1 interacted with Wnt5a to activate the NF‐κB signalling pathway through inducing phosphorylation and translocation of p65. Furthermore, Wnt5a overexpression decreased the cochlear hair cell loss. Collectively, this study suggested the protection of overexpression of ROR1 against cochlear hair cell loss in guinea pigs with NIHL via the Wnt5a‐dependent NF‐κB signalling pathway.