Transcription Factor RFX3 Stabilizes Mammary Basal Cell Identity

The myoepithelial cell compartment of the murine postnatal mammary gland is generated from basal cap cells in the terminal end bud and maintained by self-renewal. Transdifferentiation to the luminal lineage does not normally occur but can be induced by DNA damage, luminal cell death or transplantation into a recipient mammary fat pad. Myoepithelial cells cultivated in vitro can also transdifferentiate towards the luminal lineage. Little is known about the molecular mechanisms and gene regulatory networks underlying this plasticity. Using a transgenic mouse (Tg11.5kb-GFP) that marks cap cells with GFP, we discovered that mature myoepithelial cells placed in culture begin to express GFP within ~24 hrs and later express the Keratin 8 (K8) luminal marker. Cell tracking showed that most K8+ cells arose from GFP+ cells, suggesting that myoepithelial cells de-differentiate towards a progenitor state before changing lineage. Differential gene expression analysis, comparing pure GFP+ cap cells with mature myoepithelial cells, identified multiple transcription factors that iRegulon predicted might regulate the myoepithelial to cap cell transition. Knockout of one of these genes, Regulatory Factor 3 (Rfx3), significantly reduced the population of GFP+ cells and increased differentiation to the K8+ luminal lineage. Rfx3 knockout also reduced mammosphere growth and mammary gland regeneration efficiency in a transplantation assay, but had no effect on proliferation in vitro. Together, these data support a key role for Rfx3 in the stabilization of the mammary basal cell lineages.

Preservation of satellite cell number and regenerative potential with age reveals locomotory muscle bias

Background Although muscle regenerative capacity declines with age, the extent to which this is due to satellite cell-intrinsic changes vs. environmental changes has been controversial. The majority of aging studies have investigated hindlimb locomotory muscles, principally the tibialis anterior, in caged sedentary mice, where those muscles are abnormally under-exercised. Methods We analyze satellite cell numbers in 8 muscle groups representing locomotory and non-locomotory muscles in young and 2-year-old mice and perform transplantation assays of low numbers of hind limb satellite cells from young and old mice. Results We find that satellite cell density does not decline significantly by 2 years of age in most muscles, and one muscle, the masseter, shows a modest but statistically significant increase in satellite cell density with age. The tibialis anterior and extensor digitorum longus were clear exceptions, showing significant declines. We quantify self-renewal using a transplantation assay. Dose dilution revealed significant non-linearity in self-renewal above a very low threshold, suggestive of competition between satellite cells for space within the pool. Assaying within the linear range, i.e., transplanting fewer than 1000 cells, revealed no evidence of decline in cell-autonomous self-renewal or regenerative potential of 2-year-old murine satellite cells. Conclusion These data demonstrate the value of comparative muscle analysis as opposed to overreliance on locomotory muscles, which are not used physiologically in aging sedentary mice, and suggest that self-renewal impairment with age is precipitously acquired at the geriatric stage, rather than being gradual over time, as previously thought.

Absence of CD11a Expression Identifies Embryonic Hematopoietic Stem Cell Precursors via Competitive Neonatal Transplantation Assay

Hematopoietic stem cells (HSCs) are defined by their self-renewal, multipotency, and bone marrow (BM) engraftment abilities. How HSCs emerge during embryonic development remains unclear, but are thought to arise from hemogenic endothelium through an intermediate precursor called “pre-HSCs.” Pre-HSCs have self-renewal and multipotent activity, but lack BM engraftability. They can be identified functionally by transplantation into neonatal recipients, or by in vitro co-culture with cytokines and stroma followed by transplantation into adult recipients. While pre-HSCs express markers such as Kit and CD144, a precise surface marker identity for pre-HSCs has remained elusive due to the fluctuating expression of common HSC markers during embryonic development. We have previously determined that the lack of CD11a expression distinguishes HSCs in adults as well as multipotent progenitors in the embryo. Here, we use a neonatal transplantation assay to identify pre-HSC populations in the mouse embryo. We establish CD11a as a critical marker for the identification and enrichment of pre-HSCs in day 10.5 and 11.5 mouse embryos. Our proposed pre-HSC population, termed “11a- eKLS” (CD11a- Ter119- CD43+ Kit+ Sca1+ CD144+), contains all in vivo long-term engrafting embryonic progenitors. This population also displays a cell-cycle status expected of embryonic HSC precursors. Furthermore, we identify the neonatal liver as the likely source of signals that can mature pre-HSCs into BM-engraftable HSCs.

CD38 deficiency alleviates Ang II-induced vascular remodeling by inhibiting small extracellular vesicle-mediated vascular smooth muscle cell senescence in mice

CD38 is the main enzyme for nicotinamide adenine dinucleotide (NAD) degradation in mammalian cells. Decreased NAD levels are closely related to metabolic syndromes and aging-related diseases. Our study showed that CD38 deficiency significantly alleviated angiotensin II (Ang II)-induced vascular remodeling in mice, as shown by decreased blood pressures; reduced vascular media thickness, media-to-lumen ratio, and collagen deposition; and restored elastin expression. However, our bone marrow transplantation assay showed that CD38 deficiency in lymphocytes led to lack of protection against Ang II-induced vascular remodeling, suggesting that the effects of CD38 on Ang II-induced vascular remodeling might rely primarily on vascular smooth muscle cells (VSMCs), not lymphocytes. In addition, we observed that CD38 deficiency or NAD supplementation remarkably mitigated Ang II-induced vascular senescence by suppressing the biogenesis, secretion, and internalization of senescence-associated small extracellular vesicles (SA-sEVs), which facilitated the senescence of neighboring non-damaged VSMCs. Furthermore, we found that the protective effects of CD38 deficiency on VSMC senescence were related to restoration of lysosome dysfunction, particularly with respect to the maintenance of sirtuin-mediated mitochondrial homeostasis and activation of the mitochondria–lysosomal axis in VSMCs. In conclusion, our findings demonstrated that CD38 and its associated intracellular NAD decline are critical for Ang II-induced VSMC senescence and vascular remodeling.

Junctional Adhesion Molecule 2 Intensifies T Lymphopoiesis of Hematopoietic Stem Cells By Facilitating Notch/Delta Signaling

Phenotypically described hematopoietic stem cells (HSCs) represent a functionally heterogeneous pool of primitive cells with conceivable potential to replenish and maintain the whole hematopoietic system. The diverse lineage potential of HSCs is supposed to play a significant role in the response to different kinds of hematopoietic stress. Since subcategorization of HSCs biased towards specific lineage(s) highly relies on the retrospective information, e.g. transplantation assay, exploring additional markers will allow us to understand further molecular mechanisms of HSC regulation such as activation and lineage choice but also the degree of correlation between them. Here, we show that the cell surface protein Junctional adhesion molecule 2 (Jam2) serves as an amplifier of the Notch/Delta signal thereby representing the higher T cell potential of HSCs. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0%), while other Jam family member, Jam1 (F11r), was expressed on all HSCs and Jam3 was not detected. Transplantation assay using 30 Jam2+ or Jam2-HSCs revealed that Jam2+HSCs reconstituted lethally irradiated mice more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3% in peripheral blood, respectively). Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100%. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell potential of Jam2+HSCs is independent of the cell cycle progression. Jam2 has been reported to mediate the Notch signaling through an interaction with Jam1 (Kobayashi et al., Nature, 2014). In addition, Jam2+HSCs express Notch1 at a higher level than Jam2-HSCs (23.6 ±6.7 and 9.05 ±5.8%, respectively). We therefore analyzed the functional role of Jam2 in the Notch/Delta-oriented T cell production using a competitive feeder-free T cell culture system. At a low concentration of DLL1, that is insufficient to promote T cell production by itself, Jam2+HSCs effectively produced T cell lineages only in the presence of recombinant Jam1 protein, but not Jam2 or Jam3. In contrast, Jam2+HSCs did not require Jam1 protein with a higher concentration of DLL1. These differences were not observed with Jam2-HSCs, indicating that Jam2/Jam1 interaction amplifies Notch signal transduction and is crucial for the subsequent T cell specification of Jam2+HSCs. To elucidate the molecular signature of Jam2+HSCs, gene expression profiling was performed using a microarray analysis. Gene set enrichment analysis (GSEA) observed that Jam2+HSCs were significantly enriched for common lymphoid progenitor (CLP) and early T cell gene expression. Of note, Jam2+HSCs were also enriched for E2F target genes, G2M checkpoint genes and glycolysis related genes, which potentially explains the reason why Jam2+HSCs display a bivalent phenotype: being more dormant compared to Jam2-HSCs at the steady state but at the same time having the capacity to reconstitute more actively upon engraftment. Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoietic environment affects the proportion of Jam2+HSCs. In vivo T cell depletion resulted in significantly higher frequency of Jam2+HSCs but not upon other stress inducers, such as 5-FU treatment, suggesting that the increase in Jam2+HSC pool was specifically due to the T cell deficiency. These findings indicate that the lack of T cells, which also means a requirement for immediate T cell replenishment, leads to an increase of Jam2+HSC fraction. Our findings suggest that Jam2 is the key protein that controls T lymphopoiesis by enhancing the Notch/Delta signal transduction via interaction with Jam1. It also means that the lineage balance particularly towards T lymphopoiesis might be regulated at a higher stage of hematopoietic hierarchy than currently understood. Thus, Jam2 is a new marker representing the T lymphocyte potential of HSCs, as the frequency of Jam2+HSCs sensitively reflects the state of the T cell environment. Disclosures No relevant conflicts of interest to declare.

Protease-Activated Receptor 1 (PAR-1) Inhibits Proliferation but Enhances Leukemia Stem Cell Activity in Acute Myeloid Leukemia

Leukemic stem cells (LSCs) are capable of limitless self-renewal and indefinitely propagating leukemia. Eradication of LSCs is the ultimate goal of treating acute myeloid leukemia (AML). Using a mouse model of AML induced by the MLL-fusion protein MLL-AF9, we recently showed that the combined loss of Runx1/Cbfb inhibited the development of leukemia in vivo (Goyama S…Mulloy JC. Transcription factor RUNX1 promotes survival of acute myeloid leukemia cells. Journal of Clinical Investigation 123(9): 3876-3888, 2013). However, LSC-enriched cells with immature surface phenotype (cKit+Gr1-) remained viable in Runx1/Cbfb-deleted MLL-AF9 cells, indicating that RUNX targeting may not eradicate the most immature LSCs. Gene expression analyses of Runx1/Cbfb-deleted MLL-AF9 cells revealed the upregulation of thrombin pathway genes including a thrombin-activatable receptor PAR-1. Interestingly, both overexpression and knockout of PAR-1 inhibit leukemogenesis but do so through distinct mechanisms. Similar to the effect of Runx1/Cbfb-depletion, PAR-1 overexpression induced p21 expression and attenuated proliferation in MLL-AF9 cells. To our surprise, PAR-1-deficiency also prevented leukemia development induced by a small number of MLL-AF9 LSCs in vivo. Re-expression of PAR-1 in PAR-1-deficient cells combined with a limiting-dilution transplantation assay demonstrated the cell-dose dependent role of PAR-1 in MLL-AF9 leukemia: PAR-1 inhibited rapid leukemic proliferation when there are a large number of LSCs, while a small numbers of LSCs required PAR-1 for their growth. Mechanistically, PAR-1 increased adhering properties of MLL-AF9 cells and promoted their engraftment to bone marrow. PAR-1-deficiency also reduced leukemogenicity of AML1-ETO-induced leukemia. Together, these data reveal a multifaceted role for PAR-1 in leukemogenesis, and highlight this receptor as a potential target to eradicate primitive LSCs in AML. Disclosures No relevant conflicts of interest to declare.

In vivo Stem Cell Clonal Dynamics

Tremendous progress has been achieved in the characterization of the hematopoietic system over the past two decades. Historically, the main experimental approach used to elucidate and define these cellular relationships in the bone marrow (BM) has been the transplantation assay. For this reason, most of our knowledge about the in vivo properties of hematopoietic stem cells (HSCs) and progenitor cells has been derived from studies in the transplant context. Because of the lack of tractable systems, the mechanistic nature of non-transplant hematopoiesis has remained largely unexplored. Over the past several years, my laboratory has developed novel genetic tools for the clonal tracing and imaging of hematopoietic populations in the unperturbed niche that aim to bring insight into the biology of stem and progenitor cells in situ. Our work using a transposon-mediated cellular tagging approach indicated that progenitors, and not the classical long-term HSCs, are the cells mainly responsible for the day-to-day production of blood cells in the adult. Our data also suggested that lineage restricted progenitors are the main contributors to hematopoiesis at steady state. These data represent the first systematic analysis of clonal fate in an unperturbed hematopoietic niche and revealed a novel cellular mechanism for homeostatic blood regeneration. We have now utilized this clonal tracing model to bring insight into the dynamics of stem and progenitor biology during embryonic hematopoiesis and in the severely aged hematopoietic system. These data will be discussed at the meeting. Disclosures Camargo: Cell Signaling Technologies: Consultancy; Vital Therapies: Consultancy.

Establishment and Characterization of Human Germline Stem Cell Line with Unlimited Proliferation Potentials and no Tumor Formation

Spermatogonial stem cells (SSCs) have significant applications in both reproductive and regenerative medicine. However, primary human SSCs are very rare and a human SSC line has not yet been available. In this study, we have for the first time reported a stable human SSC line by stably expressing human SV40 large T antigen. RT-PCR, immunocytochemistry and Western blots revealed that this cell line was positive for a number of human spermatogonial and SSC hallmarks, including VASA, DAZL, MAGEA4, GFRA1, RET, UCHL1, GPR125, PLZF and THY1, suggesting that these cells are human SSCs phenotypically. Proliferation analysis showed that the cell line could be expanded with significant increases of cells for 1.5 years and high levels of PCNA, UCHL1 and SV40 were maintained for long-term culture. Transplantation assay indicated that human SSC line was able to colonize and proliferate in vivo in the recipient mice. Neither Y chromosome microdeletions of numerous genes nor tumor formation was observed in human SSC line although there was abnormal karyotype in this cell line. Collectively, we have established a human SSC line with unlimited proliferation potentials and no tumorgenesis, which could provide an abundant source of human SSCs for their mechanistic studies and translational medicine.