An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and several types of somatic cells. Among these, granulosa cells play a key role in follicle formation and support for oogenesis. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human induced pluripotent stem cells (hiPSCs), a method of generating granulosa cells has been elusive. Here, we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of hiPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is sufficient to generate granulosa-like cells. Our granulosa-like cells have transcriptomes similar to human fetal ovarian cells and recapitulate key ovarian phenotypes including follicle formation and steroidogenesis. When aggregated with hPGCLCs, our cells form ovary-like organoids (ovaroids) and support hPGCLC development from the premigratory to the gonadal stage as measured by induction of DAZL expression. This model system will provide unique opportunities for studying human ovarian biology and may enable the development of therapies for female reproductive health.

Ready-to-use therapeutic food (RUTF) containing less dairy may be a lower-cost treatment option for severe acute malnutrition (SAM). The objective was to understand the effectiveness of RUTF containing alternative sources of protein (nondairy), or <50% of protein from dairy products, compared with standard RUTF in children with SAM. The Cochrane Library, MEDLINE, Embase, CINAHL, and Web of Science were searched using terms relating to RUTF. Studies were eligible if they included children with SAM and evaluated RUTF with <50% of protein from dairy products compared with standard RUTF. Meta-analysis and meta-regression were completed to assess the effectiveness of intervention RUTF on a range of child outcomes. The quality of the evidence across outcomes was assessed using the GRADE (Grading of Recommendations Assessment, Development and Evaluation) approach. A total of 5868 studies were identified, of which 8 articles of 6 studies met the inclusion criteria evaluating 7 different intervention RUTF recipes. Nondairy or lower-dairy RUTF showed less weight gain (standardized mean difference: −0.20; 95% CI: −0.26, −0.15; P < 0.001), lower recovery (relative risk ratio: 0.93; 95% CI: 0.87, 1.00; P = 0.046), and lower weight-for-age z scores (WAZ) near program discharge (mean difference: −0.10; 95% CI: −0.20, 0.0; P = 0.047). Mortality, time to recovery, default (consecutive absences from outpatient therapeutic feeding program visits), nonresponse, and other anthropometric measures did not differ between groups. The certainty of evidence was high for weight gain and ranged from very low to moderate for other outcomes. RUTF with lower protein from dairy or dairy-free RUTF may not be as effective as standard RUTF for treatment of children with SAM based on weight gain, recovery, and WAZ evaluated using meta-analysis, although further research is required to explore the potential of alternative formulations. This review was registered at https://www.crd.york.ac.uk/prospero/ as CRD42020160762.

An in vitro model of human ovarian follicles would greatly benefit the study of female reproduction. Ovarian development requires the combination of germ cells and their supporting somatic cells, known as granulosa cells. Whereas efficient protocols exist for generating human primordial germ cell-like cells (hPGCLCs) from human iPSCs, a method of generating granulosa cells has been elusive. Here we report that simultaneous overexpression of two transcription factors (TFs) can direct the differentiation of human iPSCs to granulosa-like cells. We elucidate the regulatory effects of several granulosa-related TFs, and establish that overexpression of NR5A1 and either RUNX1 or RUNX2 is necessary and sufficient to generate granulosa-like cells. Our granulosa-like cells form ovary-like organoids (ovaroids) when aggregated with hPGCLCs, and recapitulate key ovarian phenotypes including support of germ cell maturation, follicle formation, and steroidogenesis. This model system will provide unique opportunities for studying human ovarian biology, and may enable the development of therapies for female reproductive health.

Treatment of acute malnutrition typically requires the provision of ready-to-use food (RUF). Common RUF is effective but expensive, being manufactured from costly ingredients, and shipped worldwide from few global suppliers. I developed a linear programming tool to create RUF optimized for low cost using locally grown crops while maintaining necessary nutritional goals and other constraints. My tool utilizes a database of the nutritional value, price, and water efficiency of suitable ingredients and allows adjustment of constraints, including nutrients, flavour, and crop water efficiency. It is designed to (a) address nutrient requirements conforming to current standards and practice; (b) optimize RUF formulae for low cost using a wide range of ingredients for nutritional value and acceptability improvement; (c) ensure protein quality through protein digestibility corrected amino acid score; and (d) adjust RUF formulae according to locally grown crop selection, local prices, and crop water footprint. The tool creates formulae free of expensive dairy ingredients, ensuring desired protein digestibility corrected amino acid score by automatically balancing proteins with complementary quantities of essential amino acids. Using publicly available data with an application to Nigeria, my tool created RUF formulae suitable for local production using local crops to meet all nutrient requirements at a fraction of the ingredient cost and water footprint of current formulae, demonstrating the tool's effectiveness. Optimization of RUF for low cost using locally grown crops will facilitate local production and reduce ingredient as well as transport costs, so more patients can receive lifesaving treatment.

Germ cells are the vehicle of human reproduction, arising early in embryonic development and developing throughout adult life until menopause onset in women. Primordial germ cells are the common precursors of germline cells in both sexes, undergoing sexual specification into oogonia or gonocytes which further develop into oocytes or spermatocytes during development. Methods for recapitulation of primordial germ cell and oogonia formation have been developed extensively in recent decades, but fundamental technical limitations in their methodologies, throughput, and yield limit their utilization. Recently, transcription factor (TF)-based methods for human primordial germ cell-like cell (hPGCLC) formation, mouse meiotic entry, and mouse oocyte maturation have demonstrated the feasibility of gene overexpression screening in identifying potent regulators of germ cell development. Here we screened 47 folliculogenesis-regulating TFs for their role in hPGCLC and oogonia formation, identifying DLX5, HHEX, and FIGLA whose individual overexpression enhances hPGCLC formation from hiPSCs. Additionally, we identify a set of three TFs, ZNF281, LHX8, and SOHLH1 whose combinatorial overexpression drives direct oogonia-like formation from hiPSCs in a four-day, feeder-free monolayer culture condition with additional feeder-free culture capabilities post-isolation. We characterize these TF-based germ cells via gene and protein expression analyses, and demonstrate their broad similarity to in vivo germ cells. Together, these results identify novel regulators of human germ cell development and establish new TF-based tools for human in vitro oogenesis research.

Objectives To develop an improved optimization tool for formulating locally suitable low-cost specialized nutritious foods (SNF) that can promote health and growth in malnourished children. Methods My method involves three parts: develop linear programming optimization tool automatically ensuring protein quality through protein digestibility-corrected amino acid score; optimize recipes according to current standards for ready-to-use therapeutic food, ready-to-use supplementary food, and super cereal plus, and compare nutrient composition and ingredient cost of optimized recipes with those of current recipes; and prepare, test, and refine a prototype super cereal plus recipe. Results My linear programming optimization tool maintains nutrient and composition constraints conforming to current standards (including the newly updated codex on protein quality in ready-to-use therapeutic food), creating low-cost recipes from local ingredients. Deriving estimates from extant data, the tool automatically ensures protein digestibility-corrected amino acid score without needing animal protein, balancing essential amino acids of ingredients. Applying the tool, I optimized SNF recipes first according to international commodity prices and next for each of 24 sub-Saharan African countries with published local price data, meeting the existing nutrient requirements, including protein quality, at minimal estimated ingredient cost, lower than ingredient cost of current recipes. Following prototyping of the optimized super cereal plus recipe, laboratory analysis verified the tool's accuracy and sensory analysis suggested appropriate acceptability. Conclusions Based on the preliminary results, my linear programming optimization tool offers an alternative, potentially effective approach to develop low-cost recipes using local crops. Funding Sources No funding was provided. Prototyping and testing of optimized super cereal plus was conducted in Kenya with the support of Valid Nutrition and Ajinomoto. Supporting Tables, Images and/or Graphs

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