The mouse mammary gland has emerged as a model system for studying processes involved in the development of epithelial tissues. Current evidence suggests the existence of a differentiation hierarchy in the mammary gland, consisting of a stem cell capable of reconstituting the tissue, progenitors with the capacity to produce specific functional cell types, and differentiated cells with limited or no repopulation potential. Although markers for mammary stem cells and progenitors have been identified, these populations have not been isolated to purity and our understanding of how they function in different stages of mammary development remains incomplete. Many adult stem cells are mitotically quiescent and can therefore retain a DNA or histone label significantly longer than differentiated cells. In an attempt to identify mammary stem cells/progenitors by histone label retention, I crossed a mouse carrying the tetracycline-inducible histone 2b/eGFP (H2BGFP) gene with tetracycline transactivator strains expected to induce H2BGFP in the mammary gland. H2BGFP expression was induced in the mammary gland until puberty and then chased for 6-8 weeks; \(H2BGFP^+\) label retaining cells were isolated and assayed. Transplantation experiments comparing MMTVrtTA/H2BGFP MECs isolated after induction to MMTVrtTA/H2BGFP MECs retaining label post-chase failed to prove that label retention enriches for stem cells/progenitors in the MMTVrtTA/H2BGFP system. During the course of these experiments, I unexpectedly discovered that MMTVrtTA induced H2BGFP expression exclusively in the \(CD24^+/CD29^+\) and \(CD24^+/CD29^{lo}\) populations, which contain stem cells and progenitors, respectively. Interestingly, I also discovered that H2BGFP+/CD24+/CD29lo MECs developed limited mammary outgrowths in vivo and that pregnancy increased the repopulation ability of these cells by 5-10-fold. H2BGFP+/CD24+/CD29lo outgrowths contained all mammary lineages and produced milk, but were unable to self-renew in serial transplant assays. Furthermore, \(H2BGFP^+/CD24^+/CD29^{lo}\) and \(H2BGFP^-/CD24^+/CD29^{lo}\) MECs had distinct gene expression profiles, with H2BGFP+/CD24+/CD29lo MECs expressing lower levels of transcripts involved in mammary development and differentiation. These data provide evidence for the existence of a multipotent, pregnancy-activated mammary progenitor and suggests that different progenitor populations are responsible for mammary expansion during puberty and pregnancy. Future studies may identify FACS markers for purification of pregnancy-activated progenitors and further elucidate the role of different mammary cell types during pregnancy.

Human induced pluripotent stem cells (iPSCs) represent a scalable cell source for the generation of hematopoietic progenitor cells (iHPCs); however, a lack of efficient iHPC expansion in vitro currently limits translational applications. To address this translational bottleneck, we assessed a panel of stem cell agonist cocktails (SCACs), originally developed to enhance cord-blood derived HSPC (CB-HSPC) expansion, on iHPC expansion. Three SCACs and GAS6 (X2A, X2A+GAS6, SM6, or SMA) were supplemented during iHPC differentiation and subsequent expansion using the STEMdiff™ Hematopoietic Kit. This monolayer differentiation strategy yielded a population of CD34⁺CD43⁺ and CD45⁺CD34⁺ iHPC. SCAC supplementation during iHPC differentiation yielded up to 2.5-fold higher frequency of CD34⁺CD43⁺ hematopoietic progenitors and up to 2.9-fold higher frequency of CD45⁺CD34⁺CD45RA⁻CD90⁺ HSC-like cells compared to non-treated controls. Subsequent SCAC supplementation during 2 weeks of expansion culture also significantly increased iHPC expansion (X2A+GAS6: 3.8-fold, X2A: 3.5-fold, SM6: 2.8-fold, SMA: 2.0-fold). The expanded iHPCs retained high levels of CD34⁺CD43⁺ expression but we observed an increase in the expansion of HSC-like cell fraction. The collective expansion observed with the SCACs was 1.5- to 2.8-fold higher than UM171 treatment alone. Furthermore, all SCAC-supplemented iHPCs retained multilineage potency, producing erythroid and granulocyte-macrophage progenitors in CFU assays. However, prolonged expansion, beyond 7 days, reduced multilineage potential, indicating a limited expansion window. Although optimal timing and composition of SCAC supplementation remains to be refined, these results highlight that exploiting the additive and synergistic effects of multiple small molecules represents a promising approach for enhancing iHPC expansion yields and biomanufacturing.