Luis A. Rodriguez

A bstract: Dynamic endocrine transitions govern female physiology, yet several reproductive hormones remain incompletely characterized across the natural menstrual cycle. Relaxin, a peptide hormone classically associated with pregnancy, has recognized vascular, extracellular matrix, and anti-fibrotic actions, but its physiology in nonpregnant women remains poorly defined. We performed dense longitudinal endocrine profiling across 80 natural menstrual cycles from 34 healthy women, complemented by oral contraceptive users, to define circulating relaxin-2 dynamics relative to ovarian steroids. Using generalized additive modeling of cycle-normalized trajectories, we identified relaxin as a highly phase-locked late-luteal hormone with a sharp, reproducible peak distinct from estradiol and progesterone. Progesterone rose earlier after ovulation and peaked at cycle day 23.9, whereas relaxin peaked significantly later at day 28.0, revealing an approximately 4-day temporal offset. Relaxin showed lower inter-cycle variability and narrower temporal dispersion than progesterone, indicating unexpectedly precise endocrine timing. Detectable relaxin persisted into early menses, suggesting continued luteal peptide release across the menstrual boundary. In combined oral contraceptive users, endogenous cyclicity was attenuated and circulating relaxin was effectively absent despite exogenous progestin exposure, implicating ovulation-dependent corpus luteum biology rather than steroid exposure alone as the primary determinant of relaxin secretion. These findings identify relaxin as a reproducible component of normal cyclic endocrine physiology rather than a hormone restricted to pregnancy. The discrete late-luteal relaxin window establishes a previously under-resolved temporal feature of the menstrual cycle and provides a framework for investigating its reproductive, vascular, and connective-tissue significance.

The results from this study have wide-ranging implications for research and treatment in women’s health including considerations in research design and interpretation for studies including women taking oral contraceptives, the potential for more precise and personalized methods of dosing to reduce unwanted side effects and adverse events, and the potential treatment of a variety of disorders ranging from musculoskeletal to neurological with exogenous hormones.

Purpose: Connexins and aquaporins play essential roles in maintaining lens homeostasis and transparency and there is a close physical and functional relationship between these two proteins. Aquaporin 0 (AQP0), in addition to its role in water transport in the lens, acts as a cell–cell adhesion molecule. Recently, we showed a new role of connexin (Cx) 50 in mediating cell–cell adhesion. However, the cooperative roles of these two proteins in the lens in vivo have not been reported. Methods: We generated an AQP0/Cx50 double knockout (dKO) mouse model. Light, fluorescence, transmission thin section, and freeze-fracture electron microscopy, as well as wheat germ agglutinin and phalloidin labeling were used to evaluate lens structure. Mechanical properties of lenses were determined by mechanical compression testing. Results: DKO mice exhibited small eyes and lenses with severe cataracts, along with lens posterior defects, including posterior capsule rupture. The dKO mouse lenses had severe structural disruption associated with increased spaces between lens fiber cells when compared with wild-type lenses or lenses deficient in either Cx50 or AQP0. DKO mice also exhibited greater reduction in lens size compared with Cx50 KO mice. Gap-junction plaque size was greatly decreased in cortical fiber cells in dKO mice. Moreover, lens stiffness and elasticity were completely diminished, exhibiting a gelatinous texture in adult dKO mice. Conclusions: This novel mouse model reveals that Cx50 and AQP0 play an important role in mediating cell–cell adhesion function in the lens fiber cells and their deficiency impairs lens fiber organization, integrity, mechanical properties, and lens development.

Mesenchymal stem cells (MSCs) are attractive cell-therapy candidates. Despite their popularity and promise, there is no uniform method of preparation of MSCs. Typically, cells are cryopreserved in liquid nitrogen, thawed, and subsequently administered to a patient with little to no information on their function post-thaw. We hypothesized that a short acclimation period post-thaw will facilitate the recovery of MSC’s functional potency. Human bone-marrow-derived MSCs were divided into 3 groups: FC (fresh cells; from existing culture); TT (thawed + time; acclimated for 24 h post-thaw); and FT (freshly thawed; thawed and immediately used). The 3 groups were analyzed for their cellular and functional potency. Phenotypic analysis demonstrated a decrease in CD44 and CD105 surface markers in FT MSCs, with no change in the other two groups. All MSCs were able to differentiate down the osteogenic and chondrogenic lineages. In FT cells, metabolic activity and apoptosis was significantly increased with concomitant decrease in cell proliferation; clonogenic capacity; and key regenerative genes. Following 24-h acclimation, apoptosis was significantly reduced in TT cells with a concomitant upregulation in angiogenic and anti-inflammatory genes. While all MSCs significantly arrested T-cell proliferation, the TT MSCs were significantly more potent. Similarly, although all MSCs maintained their anti-inflammatory properties, IFN-γ secretion was significantly diminished in FT cells. These data demonstrate that FT MSCs maintain their multipotent differentiation capacity, immunomodulatory function, and anti-inflammatory properties; yet, various aspects of cell characteristics and function are deleteriously affected by cryopreservation. Importantly, a 24-h acclimation period ‘reactivates’ thawed cells to recover their diminished stem-cell function.

Mesenchymal stem cells (MSCs) are a promising form of therapy for acute respiratory distress syndrome (ARDS). The objective of this study was two‐fold: (a) to characterize cytokine expression in serum from ARDS subjects receiving MSCs and (b) to determine MSC function following “preconditioning” with ARDS serum. In phase I, serum from three cohorts of animals (uninjured [no ARDS, n = 4], injured untreated [n = 5], and injured treated with approximately 6 million per kilogram MSCs [n = 7]) were analyzed for expression of inflammatory mediators. In phase II, the functional properties of bone marrow porcine MSCs were assessed following “preconditioning” with serum from the three cohorts. In phase III, the findings from the previous phases were validated using human bone marrow MSCs (hBM‐MSCs) and lipopolysaccharide (LPS). Serum from injured treated animals had significantly lower levels of interferon‐γ and significantly higher levels of interleukin (IL)‐1 receptor antagonist (IL‐1RA) and IL‐6. Similarly, upon exposure to the injured treated serum ex vivo, the MSCs secreted higher levels of IL‐1RA and IL‐10, dampened the secretion of proinflammatory cytokines, exhibited upregulation of toll‐like receptor 4 (TLR‐4) and vascular endothelial growth factor (VEGF) genes, and triggered a strong immunomodulatory response via prostaglandin E2 (PGE₂). hBM‐MSCs demonstrated a similar augmented therapeutic function following reconditioning in a LPS milieu. Administration of MSCs mod.ulated the inflammatory milieu following ARDS. Exposure to ARDS serum ex vivo paralleled the trends seen in vivo, which appear to be mediated, in part, through TLR‐4 and VEGF and PGE₂. Reconditioning MSCs in their own serum potentiate their immunotherapeutic function, a technique that can be used in clinical applications. Stem Cells Translational Medicine2019

Multipotent mesenchymal stromal cells (MSCs) have emerged as potent therapeutic agents for multiple indications. However, recent evidence indicates that MSC function is compromised in the physiological post-injury milieu. In this study, bone marrow (BM)- and adipose-derived (AD)-MSCs were preconditioned in hypoxia with or without inflammatory mediators to potentiate their immunotherapeutic function in preparation for in vivo delivery. Human MSCs were cultured for 48 h in either normoxia (21% O₂) or hypoxia (2% O₂) with or without the addition of Cytomix, thus creating 4 groups: (1) normoxia (21%); (2) Cytomix-normoxia (+21%); (3) hypoxia (2%); and (4) Cytomix-hypoxia (+2%). The 4 MSC groups were subjected to comprehensive evaluation of their characteristics and function. Preconditioning did not alter common MSC surface markers; nonetheless, Cytomix treatment triggered an increase in tissue factor (TF) expression. Moreover, the BM-MSCs and AD-MSCs from the +2% group were not able to differentiate to chondrocytes and osteoblasts, respectively. Following Cytomix preconditioning, the metabolism of MSCs was significantly increased while viability was decreased in AD-MSCs, but not in BM-MSCs. MSCs from both tissues showed a significant upregulation of key anti-inflammatory genes, increased secretion of IL-1 receptor antagonist (RA), and enhanced suppression of T-cell proliferation following the Cytomix treatment. Similarly, following a lipopolysaccharide challenge, the Cytomix-treated MSCs suppressed TNF-α secretion, while promoting the production of IL-10 and IL-1RA. These preconditioning approaches facilitate the production of MSCs with robust anti-inflammatory properties. AD-MSCs preconditioned with Cytomix under normoxia appear to be the most promising therapeutic candidates; however, safety concerns, such as thrombogenic disposition of cells due to TF expression, should be carefully considered prior to clinical translation.

Aim: In this study, we aimed at identifying the optimal conditions for isolation, processing and expansion of mesenchymal stem cells (MSCs). Methods: Porcine bone marrow was obtained from either sm...

In the bone marrow, MSCs reside in a hypoxic milieu (1–5% O2) that is thought to preserve their multipotent state. Typically, in vitro expansion of MSCs is performed under normoxia (~ 21% O2), a process that has been shown to impair their function. Here, we evaluated the characteristics and function of MSCs cultured under hypoxia and hypothesized that, when compared to normoxia, dedicated hypoxia will augment the functional characteristics of MSCs. Human and porcine bone marrow MSCs were obtained from fresh mononuclear cells. The first study evaluated MSC function following both long-term (10 days) and short-term (48 h) hypoxia (1% O2) culture. In our second study, we evaluated the functional characteristics of MSC cultured under short-term 2% and 5% hypoxia. MSCs were evaluated for their metabolic activity, proliferation, viability, clonogenicity, gene expression, and secretory capacity. In long-term culture, common MSC surface marker expression (CD44 and CD105) dropped under hypoxia. Additionally, in long-term culture, MSCs proliferated significantly slower and provided lower yields under hypoxia. Conversely, in short-term culture, MSCs proliferated significantly faster under hypoxia. In both long-term and short-term cultures, MSC metabolic activity was significantly higher under hypoxia. Furthermore, MSCs cultured under hypoxia had upregulated expression of VEGF with concomitant downregulation of HMGB1 and the apoptotic genes BCL-2 and CASP3. Finally, in both hypoxia cultures, the pro-inflammatory cytokine, IL-8, was suppressed, while levels of the anti-inflammatories, IL-1ra and GM-CSF, were elevated in short-term hypoxia only. In this study, we demonstrate that hypoxia augments the therapeutic characteristics of both porcine and human MSCs. Yet, short-term 2% hypoxia offers the greatest benefit overall, exemplified by the increase in proliferation, self-renewing capacity, and modulation of key genes and the inflammatory milieu as compared to normoxia. These data are important for generating robust MSCs with augmented function for clinical applications.

It is known that, following a physiological insult, bone marrow-derived mesenchymal stem cells (MSCs) mobilize and home to the site of injury. However, the effect of injury on the function of endogenous MSCs is unknown. In this study, MSCs harvested from the bone marrow of swine with or without acute respiratory distress syndrome (ARDS) were assessed for their characteristics and therapeutic function. MSCs were harvested from three groups of anesthetized and mechanically ventilated swine (n = 3 in each group): 1) no ARDS (‘Uninjured’ group); 2) ARDS induced via smoke inhalation and 40% burn and treated with inhaled epinephrine (‘Injured Treated’ group); and 3) ARDS without treatment (‘Injured Untreated’ group). Cellular evaluation of the three groups included: flow cytometry for MSC markers; colony forming unit-fibroblast (CFU-F) assay; proliferative and metabolic capacity; gene expression using quantitative real-time polymerase chain reaction (qRT-PCR); and a lipopolysaccharide (LPS) challenge, with or without coculture with mononuclear cells (MNCs), for evaluation of their protein secretion profile using Multiplex. Statistical analysis was performed using one- or two-way analysis of variance (ANOVA) with a Tukey’s post-test; a p-value less than 0.05 was considered statistically significant. Cells from all groups exhibited nearly 100% expression of MSC surface markers and retained their multidifferentiation capacity. However, the MSCs from the ‘Injured Untreated’ group generated a significantly higher number of colonies compared with the other two groups (p < 0.0001), indicative of increased clonogenic capacity following ARDS. Following an LPS challenge, the MSCs from the ‘Injured Untreated’ group exhibited a significant reduction in their proliferative capacity (p = 0.0002), significant downregulation in the expression of high-mobility group box 1 (HMGB1; p < 0.001), Toll-like receptor (TLR)-4 (p < 0.01), and vascular endothelial growth factor (VEGF; p < 0.05) genes, and significantly diminished secretory capacity for the inflammatory mediators interleukin (IL)-6 (p < 0.0001), IL-8 (p < 0.05), and tumor necrosis factor (TNF)-α (p < 0.05) compared with the ‘Uninjured’ group. The results suggest that, following ARDS, there is an increase in the clonogenic capacity of MSCs to increase the available stem cell pool in vivo. However, MSCs harvested from subjects with ARDS seem to exhibit a diminished capacity to proliferate, express regenerative signals, and secrete pro/anti-inflammatory mediators.