The expression of a battery of trophoblast-specific mRNAs was studied during trophectoderm development in vivo and in vitro to assess the use of these mRNAs as markers of trophoblast differentiation and to examine lineage relationships between various trophectoderm derivatives. In situ hybridization of sectioned day 6.5-18.5 mouse embryos localized mRNAs for mouse placental lactogens I and II and mouse proliferin (PLF) to trophoblast giant cells and proliferin-related protein mRNA to the spongiotrophoblast and giant cell layers. A fifth marker, cDNA 4311, was found only in spongiotrophoblast. Day 3.5 blastocyst outgrowths and day 7.5 diploid extraembryonic ectoderm (EX) and ectoplacental cone (EPC) were then cultured to produce polyploid giant cells in vitro. Cultures were processed for in situ hybridization after 2, 4, or 6 days. EX and EPC both formed secondary giant cells, which expressed all markers in the same sequence as was observed in vivo, and primary giant cells in blastocyst outgrowths expressed the early giant cell markers PLF and PL-I on days 4 and 6 of culture. EPC progressed through the sequence 2 days ahead of EX, indicating commitment of EPC to giant cell formation. These results suggest that EX, EPC, and primary and secondary giant cells all share in a common pathway of differentiation and that the highly ordered sequence of gene expression characteristic of this pathway occurs similarly in vivo and in vitro.
The molecular mechanisms that regulate the synthesis of the myometrial gap junction protein, connexin-43 (Cx-43), are controversial. We measured myometrial Cx-43 messenger RNA, protein and gap junction frequency, and area in myometrial samples collected from nonpregnant rats and pregnant rats at days 5, 10, 15, 17, 18, 19, 20, 21, 22, 23 (during delivery), and 1 day postpartum and correlated these data with plasma concentrations of estradiol 17 beta and progesterone. Cx-43 transcripts were low or undetectable (connexin-43:glyceraldehyde phosphate dehydrogenase ratio < 0.2) in nonpregnant rats or in rats before day 10 of pregnancy. Transcripts rose to 0.52 +/- 0.11 on day 10, increased (2.9-fold) to 1.51 +/- 0.48 on day 22, and increased a further 2.9-fold to maximal levels of 4.42 +/- 0.67 during labor. Cx-43 protein was barely detectable on day 21 [0.12 +/- 0.04 relative optical density (ROD) units], increased 2.5-fold on day 22 (0.30 +/- 0.04 ROD units), and a further 3.7-fold during delivery (1.10 +/- 0.15 ROD units), at a time when gap junctions were present in large numbers in the cell membrane. Between day 21 and delivery the increase in Cx-43 transcripts (8.2-fold) and protein (9.2-fold) were of a similar magnitude. There was a significant positive correlation between the increases in Cx-43 transcripts and the increase in the ratio of plasma estradiol to progesterone. Levels of Cx-43 transcripts, protein, and gap junctions fell rapidly postpartum. Our data demonstrate: 1) that transcripts encoding the gap junction protein, Cx-43, are at maximal levels during delivery and that this increase is temporally associated with increases in Cx-43 protein and the appearance of gap junctions; and 2) that these data, in association with changes in plasma steroid concentrations, are consistent with myometrial Cx-43 transcript levels being regulated positively by estrogen and negatively by progesterone during pregnancy.
Cell-based therapy is a major focus for treatment of stress urinary incontinence (SUI). However, derivation of primary cells requires tissue biopsies, which often have adverse effects on patients. A recent study used human induced pluripotent stem cells (iPSC)-derived smooth muscle myocytes for urethral sphincter regeneration in rats. Here, we establish a workflow using iPSC-derived fibroblasts and skeletal myocytes for urethral tissue regeneration: (1) Cells from voided urine of women were reprogrammed into iPSC. (2) The iPSC line U1 and hESC line H9 (control) were differentiated into fibroblasts expressing FSP1, TE7, vinculin, vimentin, αSMA, fibronectin and paxillin. (3) Myogenic differentiation of U1 and H9 was induced by small molecule CHIR99021 and confirmed by protein expression of myogenic factors PAX7, MYOD, MYOG, and MF20. Striated muscle cells enriched by FACS expressed NCAM1, TITIN, DESMIN, TNNT3. (4) Human iPSC-derived fibroblasts and myocytes were engrafted into the periurethral region of RNU rats. Injected cells were labelled with ferric nanoparticles and traced by Prussian Blue stain, human-specific nuclear protein KU80, and human anti-mitochondria antibody. This workflow allows the scalable derivation, culture, and in vivo tracing of patient-specific fibroblasts and myocytes, which can be assessed in rat SUI models to regenerate urethral damages and restore continence.
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