Spermatogonial stem cells (SSCs) sustain spermatogenesis by balancing self-renewal and initiation of differentiation to produce progenitor spermatogonia committed to forming sperm. To define the regulatory logic among SSCs and progenitors, we performed single-cell RNA velocity analyses and validated results in vivo. A predominant quiescent SSC population spawns a small subset of cell-cycle-activated SSCs via mitogen-activated protein kinase (MAPK)/AKT signaling. Activated SSCs form early progenitors and mTORC1 inhibition drives activated SSC accumulation consistent with blockade to progenitor formation. Mechanistically, mTORC1 inhibition suppresses transcription among spermatogonia and specifically alters expression of insulin growth factor (IGF) signaling in early progenitors. Tex14-/- testes lacking intercellular bridges do not accumulate activated SSCs following mTORC1 inhibition, indicating that steady-state mTORC1 signaling drives activated SSCs to produce progenitor clones. These results are consistent with a model of SSC self-renewal dependent on interconversion between activated and quiescent SSCs, and mTORC1-dependent initiation of differentiation from SSCs to progenitor clones.
Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.

Spermatogenesis is a complex and dynamic cellular differentiation process critical to male reproduction and sustained by spermatogonial stem cells (SSCs). Although patterns of gene expression have been described for aggregates of certain spermatogenic cell types, the full continuum of gene expression patterns underlying ongoing spermatogenesis in steady state was previously unclear. Here, we catalog single-cell transcriptomes for >62,000 individual spermatogenic cells from immature (postnatal day 6) and adult male mice and adult men. This allowed us to resolve SSC and progenitor spermatogonia, elucidate the full range of gene expression changes during male meiosis and spermiogenesis, and derive unique gene expression signatures for multiple mouse and human spermatogenic cell types and/or subtypes. These transcriptome datasets provide an information-rich resource for studies of SSCs, male meiosis, testicular cancer, male infertility, or contraceptive development, as well as a gene expression roadmap to be emulated in efforts to achieve spermatogenesis in vitro.
Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Vandetanib is an orally active small molecule tyrosine kinase inhibitor (TKI) with activity against several pathways implicated in malignancy including the vascular endothelial growth factor receptor pathway, the epidermal growth factor receptor pathway, the platelet derived growth factor receptor β pathway, and REarranged during Transfection pathway. To determine if vandetanib-mediated inhibition of receptor tyrosine kinases is a potential therapeutic strategy for pediatric acute leukemia, these studies aimed to characterize the activity of vandetanib against acute leukemia in vitro. Treatment of leukemia cell lines with vandetanib resulted in a dose-dependent decrease in proliferation and survival. Vandetanib's anti-leukemic activity appeared mediated by multiple mechanisms including accumulation in G1 phase at lower concentrations and apoptosis at higher concentrations. Alterations in cell surface markers also occurred with vandetanib treatment, suggesting induction of differentiation. In combination with DNA damaging agents (etoposide and doxorubicin) vandetanib demonstrated synergistic induction of cell death. However in combination with the anti-metabolite methotrexate, vandetanib had an antagonistic effect on cell death. Although several targets of vandetanib are expressed on acute leukemia cell lines, expression of vandetanib targets did not predict vandetanib sensitivity and alone are therefore not likely candidate biomarkers in patients with acute leukemia. Interactions between vandetanib and standard chemotherapy agents in vitro may help guide choice of combination regimens for further evaluation in the clinical setting for patients with relapsed/refractory acute leukemia. Taken together, these preclinical data support clinical evaluation of vandetanib, in combination with cytotoxic chemotherapy, for pediatric leukemia.

Vascularization is the cornerstone of wound healing. We introduced human blood outgrowth endothelial cells (hBOEC) in a self-assembled human dermal fibroblast sheet (hDFS), intended as a tissue-engineered dermal substitute with inherent vascular potential. hBOEC were functionally and molecularly different from early endothelial progenitor cells and human umbilical vein endothelial cells (HUVEC). hBOEC alone, unlike HUVEC, efficiently revascularized and re-oxygenated the wound bed, both by active incorporation into new vessels and by trophic stimulation of host angiogenesis in a dose-dependent manner. Furthermore, hBOEC alone, but not HUVEC, accelerated epithelial coverage and matrix organization of the wound bed. In addition, integration of hBOEC in hDFS not only further improved vascularization, epithelial coverage and matrix organization but also prevented excessive wound contraction. In vitro analyses with hBOEC, fibroblasts and keratinocytes revealed that these effects were both due to growth factor crosstalk and to short cutting hypoxia. Among multiple growth factors secreted by hBOEC, placental growth factor mediated at least in part the beneficial effects on keratinocyte migration and proliferation. Overall, this combined tissue engineering approach paves the way for clinical development of a fully autologous vascularized dermal substitute for patients with large skin defects that do not heal properly.

Polyclonal B cell activation is a well-described feature of systemic lupus erythematosus (SLE), but the immune mechanisms leading to this activation are unclear. To gain insight into these processes, we extensively characterized the activated peripheral blood B cell populations in SLE. PBMC from lupus patients and healthy controls were stained with various combinations of conjugated Ab to identify distinct peripheral B cell subsets, and activation was assessed by measurement of forward scatter and CD80 or CD86 expression using flow cytometry. SLE patients had altered proportions of several B cell subsets, many of which demonstrated increased activation as assessed by forward scatter. This activation occurred at an early developmental stage, as B cells in the transitional (T2) stage were already significantly larger than those seen in controls. Increased proportions of CD80- or CD86-expressing cells were also seen in multiple B cell subsets, with the most striking differences observed in the naive CD27-CD23+ population. Within the CD23+ subset, increased costimulatory molecule expression was most pronounced in an IgD+IgMlow population, suggesting that activation follows Ag engagement. Although controls also had IgD+IgMlowCD23+ cells, they were reduced in number and not activated. Thus, there is an altered response to Ig receptor engagement with self-Ags in lupus.