Biology subjects

Koganti, P. P.

Publications and source records attributed to Koganti, P. P..

4 recordsLinked to original sources

bioRxiv2025

From blood to pluripotency: Fibrocytes as a reprogrammable somatic cell source for bovine iPSCs

Fibrocytes represent a distinct somatic cell type derived from peripheral blood leukocytes, first described as spindle-shaped adherent cells with dual hematopoietic and mesenchymal features. Although fibrocytes were identified in early descriptive studies across several mammalian systems, most of this work predated modern molecular approaches, and the cells remain incompletely defined at the molecular level and have not previously been derived or characterized in cattle. Seeking somatic cells that could be collected aseptically and reproducibly under field conditions for reprogramming to pluripotency, we recognized fibrocytes as a practical and previously unexplored candidate population. Here, we establish a reproducible method for fibrocyte derivation and expansion from adult bovine blood and define their molecular identity using transcriptomic and network analyses. Principal component and differential expression analyses revealed extensive immune, inflammatory, metabolic, and stress-responsive pathways that distinguished fibrocytes from fibroblasts. Upstream regulator analysis identified a fibrocyte-restricted transcriptional network governed by SPI1, IRF5/IRF7, NFKBIZ, PRDM1, CIITA, and MAFB, supporting a monocyte-derived origin and indicating some retention of hematopoietic lineage memory despite acquisition of mesenchymal features. Optimized fibrocyte medium (FbC; dexamethasone, ascorbate, PDGF-BB, EGF, A83-01, CHIR99021) supported stable proliferation and selectively enhanced cytoskeletal and matrix-constructive programs while attenuating inflammatory tone. When reprogrammed with polycistronic OCT4-SOX2-KLF4-cMYC and SV40 large T antigen, fibrocytes generated induced pluripotent stem cell (iPSC) colonies exhibiting defining molecular and morphological features of pluripotency. These findings establish fibrocytes as a field-adaptable, stably expandable, and reprogrammable somatic cell type with practical applications in induced pluripotent stem cell generation, genetic preservation, and reproductive biotechnology.

cell biology↗

bioRxiv2025

Flexible oocyte manipulation with delayed maturation and improved SCNT efficiency using induced pluripotent stem cells

Somatic cell nuclear transfer (SCNT) remains inefficient, limiting its practical use in cattle reproduction and research. This study investigated two complementary strategies to enhance handmade cloning (HMC): (1) holding bovine oocytes overnight and delaying maturation to enable a second round of SCNT and (2) using bovine-induced pluripotent stem cells (biPSCs) as donor nuclei to enhance developmental competence. Bovine oocytes were subjected to either conventional in vitro maturation (CONV; 20 h) or delayed maturation using a holding medium for 20 h before CONV (HOLD). Matured oocytes were used for SCNT, parthenogenetic activation (PA), or in vitro fertilization (IVF) as controls. Handmade SCNT embryos were reconstructed using fibroblasts or biPSCs as donors, activated, and cultured for 7 days. Results showed no significant differences between CONV and HOLD groups in oocyte maturation, recovery after stripping, survival after zona removal, or cleavage and blastocyst development after SCNT. Fusion rates using fibroblasts were comparable between groups (42.6{+/-}6.0% vs. 50.3{+/-}9.8%), with biPSCs showing significantly higher fusion rates in CONV group (85.7{+/-}8.2% vs. 50.5{+/-}8.8%, P<0.05). Among fused embryos, biPSCs produced higher blastocyst rates (33.3{+/-}16.7%) compared with fibroblast donors (21.9{+/-}12.6%, P<0.05). Across all reconstructed embryos, cleavage and blastocyst development were also greater with biPSCs (odds ratios 3.4 and 2.7 respectively). These findings indicate that delaying maturation offers flexible timing for SCNT without compromising competence. Moreover, biPSCs enhance embryo developmental outcomes, supporting their use as superior donor cells for advancing cloning efficiency and applications in reproductive biotechnology.

developmental biology↗

bioRxiv2025

Target validation uncouples TSPO from 19-Atriol-mediated inhibition of steroidogenesis and reveals true enzymatic targets

The mitochondrial translocator protein (TSPO) was once proposed to mediate mitochondrial cholesterol import for steroid hormone biosynthesis, but genetic deletion studies in multiple models have refuted this role. Nevertheless, the idea that pharmacological ligands of TSPO can modulate steroid output continues to be invoked. One such compound, 19-Atriol (androst-5-ene-3{beta},17{beta},19-triol), was reported to inhibit progesterone synthesis via TSPO binding in MA-10 Leydig cells. To evaluate this proposed mechanism, we used CRISPR/Cas9-generated Tspo-deleted MA-10 cells to study 19-Atriol activity. We found that 19-Atriol inhibited Bt2-cAMP-stimulated steroid output independent of TSPO expression; it acted as a competitive inhibitor of 3{beta}-hydroxysteroid dehydrogenase (3{beta}-HSD), blocking the conversion of pregnenolone to progesterone. Mass spectrometry revealed that 19-Atriol is also a substrate for 3{beta}-HSD, yielding 19-hydroxytestosterone (19-OHT), which itself inhibits 3{beta}-HSD activity. In addition to this effect, both 19-Atriol and 19-OHT decreased cholesterol-to-pregnenolone conversion during stimulation. Partial inhibition of 22R-hydroxycholesterol metabolism by CYP11A1 was observed with 19-Atriol, but not 19-OHT, suggesting direct or indirect effects on this upstream step, potentially involving the steroidogenic acute regulatory protein (STAR). These findings decisively exclude TSPO as a functional mediator of 19-Atriol activity and instead identify direct enzymatic targets within the de novo steroidogenic pathway. By resolving a key mechanistic misattribution, this study underscores the importance of rigorous target validation, particularly for compounds previously assumed to act via TSPO.

pharmacology and toxicology↗

bioRxiv2025

STAR/STARD1: a mitochondrial intermembrane space cholesterol shuttle degraded through mitophagy

The import of cholesterol to the inner mitochondrial membrane by the steroidogenic acute regulatory protein (STAR/STARD1) is essential for de novo steroid hormone biosynthesis and the acidic pathway of bile acid synthesis. This robust system, evolved to start and stop colossal cholesterol movement, ensures pulsatile yet swift mitochondrial steroid metabolism in cells. Nonetheless, the proposed mechanism and components involved in this process has remained a topic of ongoing debate. In this study, we elucidate the mitochondrial import machinery and structural aspects of STAR, revealing its role as an intermembrane space cholesterol shuttle that subsequently undergoes rapid degradation by mitophagy. This newfound mechanism illuminates a fundamental process in cell biology and provides precise interpretations for the full range of human STAR mutation-driven lipoid congenital adrenal hyperplasia in patients. One Sentence SummarySTAR activates mitochondrial steroid metabolism as a cholesterol shuttle in the intermembrane space and is destroyed by mitophagy.

cell biology↗