Bedford Research Foundation

Recently, a mixture of hemoglobin (Hb) and bovine serum albumin (BSA) was separated into two highly purified fractions with high recovery via internally staged ultrafiltration (ISUF) having a stack of three identical flat 100 kDa ultrafiltration (UF) membranes in a stirred cell. Selectivities as high as 1000–4000+, and high recoveries of individual species were achieved. Successful separation and purification of IgG from a BSA-IgG mixture was also achieved in a stirred cell using a modified ISUF technique with two 100 kDa membranes on a 70 kDa membrane; here BSA was a model for host cell proteins (HCPs) in post-protein A eluate. We explored here separation and purification of the Hb-BSA system in a developmental cassette of 88 cm 2 membrane surface area, having a stack of three 100 kDa membranes on each side of the channel. The channel gap was smaller than that in regular cassettes resulting in higher flow pressure drops. The Hb-BSA separation was studied over a diavolume (DV) range of ∼0–6. The Hb-BSA selectivity was as high as 1600 and remained above 300 till 6 DV. Lower feed pressure and a particular permeate collection mode leading to reduced transmembrane pressure drop were crucial for higher performance. Species recoveries were lower for the DV range used especially compared to those in a stirred cell. Successful separation and purification of BSA-IgG system was also demonstrated with similar limitations. Increased flow pressure drop due to flow channel height reduction in existing cassette frames and lower flow rates leading to higher concentration polarization suggest modification of the cassette frame for high performance ISUF for difficult-to-separate protein mixtures in biopharmaceutical separation processes. • Internally staged ultrafiltration (ISUF) separates difficult binary protein mixtures. • Each channel side in a scaled-up ISUF cassette had a three 100 kDa membrane stack. • Cassette selectivity of Hb-BSA was a high of 1600 and remained >300 till 6 diavolume. • A modified ISUF stack can yield very high cassette performance for BSA-IgG system. • Larger channel gap to reduce flow pressure drop will enhance system performances.

Abstract Human induced pluripotent stem cells (hiPSCs) show great promise in the development of novel strategies to mitigate reproductive diseases and promote successful reproductive outcomes. Recently, a novel approach for the fast and efficient differentiation of ovarian support cells (OSCs) to generate a versatile platform for basic research and clinical applications was demonstrated. This study details the clinical process development and application of an OSC product, known asFertilo, to improve thein vitromaturation (IVM) of human oocytes, a method referred to as OSC-IVM. First, transcription factor (TF) mediated OSC differentiation using research-grade raw materials was shown to produce granulosa-like cells that improve the MII maturation rate of human oocytes. To support clinical application, several raw material upgrades were initiated, including substitution of the differentiation matrix with a higher-quality alternative, laminin-521, and the generation of a clinically suitable hiPSC seed bank and master cell bank. Single cell RNA sequencing of OSCs generated using the updated protocol for clinical translation demonstrated the consistency and reproducibility of cellular outcomes. Next, analytical release testing of the clinical product was performed and a murine oocyte maturation assay was developed to establish the potency of OSCs for use in OSC-IVM. Finally, the qualifiedFertiloproduct was applied in a two-phase longitudinal cohort analysis, with the results showing improvement in key outcomes compared to traditional IVM treatment. Our findings demonstrate the first-time clinical development and application of an hiPSC-derived product to improve reproductive outcomes after IVM and advance women’s health.

This paper endeavors to scrutinize methodologies and approaches geared towards ameliorating the developer experience with a specific emphasis on strategies aimed at curtailing cognitive load. By delving into minimization strategies, the study seeks to unravel intricacies associated with mental effort reduction in software development tasks. The ultimate objective is to provide nuanced insights that can positively influence the cognitive dimensions impacting developers, ultimately enhancing productivity, decision-making, and the overall cognitive ergonomics of software development practices. The outcomes of this paper contribute substantively to the ongoing discourse surrounding optimization paradigms for developers, fostering a technologically advanced and cognitively attuned software development milieu.

This study is to discover hormone pathways active in early cleaving human embryos. A list of 152 hormones and receptors were compiled to query the microarray database of mRNAs in 8-cell human embryos, two lines of human embryonic stem cells plus human fibroblasts before and after induced pluripotency. Over half of the 152 hormones and receptors were silent on the arrays of all cell types, and more were detected at high or moderate levels on the 8-cell arrays than on the pluripotent cell or fibroblast arrays. Eight hormone family genes were uniquely detected at least 22-fold higher on the 8-cell arrays than the stem cell arrays: AVPI1, CCK, CORT, FSTL4, GIP, GPHA2, OXT, and PPY suggesting novel roles for these proteins in early development. Oxytocin was detected by pilot immunoassay in culture media collected from Day 3 embryos. Robust detection of CRHR1 and EPOR suggests the 8-cell embryo may be responsive to maternal CRH and EPO. The over-expression of POMC and GHITM suggests POMP peptide products may have undiscovered roles in early development and GHITM may contribute to mitochondrial remodeling. Under-detected on the 8-cell arrays at least tenfold were two key enzymes in steroid biosynthesis, DHCR24 and FDPS. The 8-cell human embryo may be secreting oxytocin, which could stimulate its own progress down the fallopian tube as well as play a role in early neural precursor development. The 8-cell embryo does not synthesize reproductive steroid hormones. As previously reported for growth factor families, the early embryo over-expresses more hormones than hormone receptors.