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We evaluated the engraftment and the cell cycle status of marrow cells at various times after 5-fluorouracil (5-FU) administration. 5-FU (150 mg/kg) was given to donor male BALB/c mice at 1, 2, 6, or 12 days prior to marrow harvest. The donor cells were then assessed in host nonmyeloablated female mice. Bone marrow engraftment of marrow treated with 5-FU was evaluated and compared to marrow treated with diluent (phosphate-buffered saline) at 3 and 10 weeks after marrow infusion. Our data show a rapid induction of an engraftment defect 1 day after 5-FU, persistence of this defect through day 6, and a recovery by day 12. Experiments using hydroxyurea (which selectively kills cells in the S phase) to determine the cell cycle status indicated that cells that engrafted in post-5-FU marrow were noncycling at days 1, 2, and 12 but cycling at day 6. Post-5-FU bone marrow was also analyzed in vitro by colony assays and its cycling status determined by 3H-thymidine suicide assay. High-proliferative-potential colony-forming cells (HPP-CFCs) and low-proliferative-potential colony-forming cells (LPP-CFCs) decreased rapidly 1 day after 5-FU, with a nadir observed at day 6 for HPP-CFCs and day 2 for LPP-CFCs. By day 12, LPP-CFCs showed a total recovery, but HPP-CFCs were still defective. Significant numbers of HPP-CFCs were cycling, mostly at days 6 and 8 after 5-FU, whereas LPP-CFCs appeared quiescent except at day 2. These results emphasize the importance of timing if post-5-FU marrow is used for gene therapy or marrow transplantation. [ABSTRACT FROM AUTHOR]

Historically hematopoietic stem cells are believed to be predominantly dormant but could be induced into active cell cycle under specific conditions. This review, coupled with years of research from our laboratory, challenges this belief by demonstrating a significant portion of hematopoietic stem cells are actively cycling rather than quiescent. This addresses a major heuristic error in the understanding of hematopoietic stem cells that has shaped this field for decades. By evaluating the cycle status of engraftable hematopoietic stem cells in whole unseparated bone marrow, we demonstrated that a significant portion of these cells are actively cycling, and further confirmed by tritiated thymidine suicide and bromodeoxyuridine labeling assays. Moreover, by analyzing both whole unseparated bone marrow and purified lineage-negative hematopoietic stem cells in murine models, our findings indicate that lineage-positive cells, usually discarded during purification, actually contain actively cycling stem cells. Taken together, our findings highlight that hematopoietic stem cells are characterized as actively cycling and expressing differentiation epitopes. This corrects a basic mistake in stem cell biology. Furthermore, these findings provide valuable insights for a better understanding of the actively cycling hematopoietic stem cells in the field of stem cell biology.

Glycogen synthase kinase-3 (GSK-3) is a serine/threonine kinase that has been implicated in numerous oncogenic processes. GSK-3 inhibitor elraglusib (9-ING-41) has shown promising preclinical and clinical antitumor activity across multiple tumor types. Despite promising early-phase clinical trial results, there have been limited efforts to characterize the potential immunomodulatory properties of elraglusib. We report that elraglusib promotes immune cell-mediated tumor cell killing of microsatellite stable colorectal cancer (CRC) cells. Mechanistically, elraglusib sensitized CRC cells to immune-mediated cytotoxicity and enhanced immune cell effector function. Using western blots, we found that elraglusib decreased CRC cell expression of NF-κB p65 and several survival proteins. Using microarrays, we discovered that elraglusib upregulated the expression of proapoptotic and antiproliferative genes and downregulated the expression of cell proliferation, cell cycle progression, metastasis, TGFβ signaling, and anti-apoptotic genes in CRC cells. Elraglusib reduced CRC cell production of immunosuppressive molecules such as VEGF, GDF-15, and sPD-L1. Elraglusib increased immune cell IFN-γ secretion, which upregulated CRC cell gasdermin B expression to potentially enhance pyroptosis. Elraglusib enhanced immune effector function resulting in augmented granzyme B, IFN-γ, TNF-α, and TRAIL production. Using a syngeneic, immunocompetent murine model of microsatellite stable CRC, we evaluated elraglusib as a single agent or combined with immune checkpoint blockade (anti-PD-1/L1) and observed improved survival in the elraglusib and anti-PD-L1 group. Murine responders had increased tumor-infiltrating T cells, augmented granzyme B expression, and fewer regulatory T cells. Murine responders had reduced immunosuppressive (VEGF, VEGFR2) and elevated immunostimulatory (GM-CSF, IL-12p70) cytokine plasma concentrations. To determine the clinical significance, we then utilized elraglusib-treated patient plasma samples and found that reduced VEGF and BAFF and elevated IL-1 beta, CCL22, and CCL4 concentrations correlated with improved survival. Using paired tumor biopsies, we found that tumor-infiltrating immune cells had a reduced expression of inhibitory immune checkpoints (VISTA, PD-1, PD-L2) and an elevated expression of T-cell activation markers (CTLA-4, OX40L) after elraglusib treatment. These results address a significant gap in knowledge concerning the immunomodulatory mechanisms of GSK-3 inhibitor elraglusib, provide a rationale for the clinical evaluation of elraglusib in combination with immune checkpoint blockade, and are expected to have an impact on additional tumor types, besides CRC., Competing Interests: Elraglusib (9-ING-41) has been licensed to Actuate Therapeutics. K.E.H., P.S., and W.S.E-D. receive research funding for preclinical studies from Actuate Therapeutics, Inc (March 2022- Feb. 2024). B.A.C. received institutional funding for a clinical trial related to 9-ING-41 from Actuate Therapeutics, Inc. F.J.G. has served as a consultant to Actuate Therapeutics, Inc. Daniel Newhouse is an employee and shareholder of NanoString Technologies Inc. All remaining authors report no disclosures.

Meniscal tearing in the knee increases the risk of post-traumatic osteoarthritis (OA) in patients. The therapeutic application of tissue-specific mesenchymal progenitor cells is currently being investigated as an emerging biologic strategy to help improve healing of musculoskeletal tissues like meniscal fibrocartilage and articular hyaline cartilage. However, many of these approaches involve isolating cells from healthy tissues, and the low yield of rare progenitor populations (< 1% of total cells residing in tissues) can make finding a readily available cell source for therapeutic use a significant logistical challenge. In the present study, we investigated the therapeutic efficacy of using expanded cartilage-derived and bone marrow-derived progenitor cell lines, which were stabilized using retroviral SV40, for repair of meniscus injury in a rodent model. Our findings indicate that these cell lines express the same cell surface marker phenotype of primary cells (CD54+, CD90+, CD105+, CD166+), and that they exhibit improved proliferative capacity that is suitable for extensive expansion. Skeletally mature male athymic rats treated with 3.2 million cartilage-derived progenitor cell line exhibited approximately 79% greater meniscal tear reintegration/healing, compared to injured animals that left untreated, and 76% greater compared to animals treated with the same number of marrow-derived stromal cells. Histological analysis of articular surfaces also showed that cartilage-derived progenitor cell line treated animals exhibited reduced post-traumatic OA associated articular cartilage degeneration. Stable cell line treatment did not cause tumor formation or off-target engraftment in animals. Taken together, we present a proof-of-concept study demonstrating, for the first time, that intra-articular injection of a stable human cartilage-derived progenitor cell line stimulates meniscus tear healing and provide chondroprotection in an animal model. These outcomes suggest that the use of stable cell lines may help overcome cell source limitations for cell-based medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Desai, Dooner, Newberry, Twomey-Kozak, Molino, Trivedi, Patel, Owens and Jayasuriya.)

Traumatic brain injury (TBI) is of significant concern in the realm of high impact contact sports, including mixed martial arts (MMA). Extracellular vesicles (EVs) travel between the brain and oral cavity and may be isolated from salivary samples as a noninvasive biomarker of TBI. Salivary EVs may highlight acute neurocognitive or neuropathological changes, which may be particularly useful as a biomarker in high impact sports. Pre and post-fight samples of saliva were isolated from 8 MMA fighters and 7 from controls. Real-time PCR of salivary EVs was done using the TaqMan Human Inflammatory array. Gene expression profiles were compared pre-fight to post-fight as well as pre-fight to controls. Largest signals were noted for fighters sustaining a loss by technical knockout (higher impact mechanism of injury) or a full match culminating in referee decision (longer length of fight), while smaller signals were noted for fighters winning by joint or choke submission (lower impact mechanism as well as less time). A correlation was observed between absolute gene information signals and fight related markers of head injury severity. Gene expression was also significantly different in MMA fighters pre-fight compared to controls. Our findings suggest that salivary EVs as a potential biomarker in the acute period following head injury to identify injury severity and can help elucidate pathophysiological processes involved in TBI.

Rationale: Sex hormones play a role in pulmonary arterial hypertension (PAH), but the menstrual cycle has never been studied. Objectives: We conducted a prospective observational study of eight women with stable PAH and 20 healthy controls over one cycle. Methods: Participants completed four study visits 1 week apart starting on the first day of menstruation. Relationships between sex hormones, hormone metabolites, and extracellular vesicle microRNA (miRNA) expression and clinical markers were compared with generalized linear mixed modeling. Results: Women with PAH had higher but less variable estradiol (E2) levels ( P  < 0.001) that tracked with 6-minute walk distance ( P  < 0.001), N-terminal prohormone of brain natriuretic peptide ( P  = 0.03) levels, and tricuspid annular plane systolic excursion ( P  < 0.01); the direction of these associations depended on menstrual phase. Dehydroepiandrosterone sulfate (DHEA-S) levels were lower in women with PAH (all visits, P  < 0.001). In PAH, each 100-μg/dl increase in DHEA-S was associated with a 127-m increase in 6-minute walk distance ( P  < 0.001) and was moderated by the cardioprotective E2 metabolite 2-methoxyestrone ( P  < 0.001). As DHEA-S increased, N-terminal prohormone of brain natriuretic peptide levels decreased ( P  = 0.001). Expression of extracellular vesicle miRNAs-21, -29c, and -376a was higher in PAH, moderated by E2 and DHEA-S levels, and tracked with hormone-associated changes in clinical measures. Conclusions: Women with PAH have fluctuations in cardiopulmonary function during menstruation driven by E2 and DHEA-S. These hormones in turn influence transcription of extracellular vesicle miRNAs implicated in the pathobiology of pulmonary vascular disease and cancer.