Matrix Gla protein (MGP) is a phosphorylated and γ-carboxylated protein that has been shown to prevent the deposition of hydroxyapatite crystals in the walls of blood vessels. MGP is also expressed in kidney and may inhibit the formation of kidney stones, which mainly consist of another crystalline phase, calcium oxalate monohydrate. To determine the mechanism by which MGP prevents soft-tissue calcification, we have synthesized peptides corresponding to the phosphorylated and γ-carboxylated sequences of human MGP in both post-translationally modified and non-modified forms. The effects of these peptides on hydroxyapatite formation and calcium oxalate crystallization were quantified using dynamic light scattering and scanning electron microscopy, respectively. Peptides YGlapS (MGP1-14: YγEpSHEpSMEpSYELNP), YEpS (YEpSHEpSMEpSYELNP), YGlaS (YγESHESMESYELNP) and SK-Gla (MGP43-56: SKPVHγELNRγEACDD) inhibited formation of hydroxyapatite in order of potency YGlapS > YEpS > YGlaS > SK-Gla. The effects of YGlapS, YEpS and YGlaS on hydroxyapatite formation were on both crystal nucleation and growth; the effect of SK-Gla was on nucleation. YGlapS and YEpS significantly inhibited the growth of calcium oxalate monohydrate crystals, while simultaneously promoting the formation of calcium oxalate dihydrate. The effects of these phosphopeptides on calcium oxalate monohydrate formation were on growth of crystals rather than nucleation. We have shown that the use of dynamic light scattering allows inhibitors of hydroxyapatite nucleation and growth to be distinguished. We have also demonstrated for the first time that MGP peptides inhibit the formation of calcium oxalate monohydrate. Based on the latter finding, we propose that MGP function not only to prevent blood-vessel calcification but also to inhibit stone formation in kidney.

Human PSC (hPSC) differentiations can capture developmental phenotypes and processes and are useful for studying fundamental biological mechanisms driving tissue morphogenesis and cell lineage development. Here, we show for the first time the temporal development of lung cell lineages using hPSC that model developmental milestones observed in primary tissue, the generation of renewable fetal lung epithelial organoids, and the functional utility of the lung models at different differentiation stages for Cystic fibrosis disease modeling. We first show the presence of hPSC-derived lung progenitor cells reminiscent of early trimester lung development and can capture a population enriched with basal stem cells that generates renewable airway organoids. Maturation and polarization in air liquid interface (ALI) generates additional epithelial cell lineages found in adult lung tissues including pulmonary neuroendocrine, brush, mature basal, ciliated and secretory cell types. Finally, pseudotime analysis of the integrated datasets from the fetal and ALI stages reveal the developmental trajectories of the cells as they emerge during differentiation. Overall, hPSC differentiation can capture aspects of human lung development and potentially provide important insight into congenital causes of diseases.

Hedgehog signaling is essential for vertebrate development, however, less is known about the negative regulators that influence this pathway during the differentiation of cell fates. Using the mouse P19 embryonal carcinoma cell model, Suppressor of Fused (SUFU), a negative regulator of the Hedgehog pathway, was investigated during retinoic acid-induced neural differentiation. We found Hedgehog signaling was activated in the early phase of neural differentiation and became inactive during terminal differentiation of neurons and astrocytes. SUFU, which regulates signaling at the level of GLI, remained relatively unchanged during the differentiation process, however SUFU loss through CRISPIR-Cas9 gene editing resulted in decreased cell proliferation and ectopic expression of Hedgehog target genes. Interestingly, SUFU-deficient cells were unable to differentiate in the absence of retinoic acid, but when differentiated in its presence they showed delayed and decreased astrocyte differentiation; neuron differentiation did not appear to be affected. Retinoic acid-induced differentiation also caused ectopic activation of Hh target genes in SUFU-deficient cells and while the absence of the GLI3 transcriptional inhibitor suggested the pathway was active, no full-length GLI3 was detected even though the message encoding Gli3 was present. Thus, the study would indicate the proper timing and proportion of glial cell differentiation requires SUFU, and its normal regulation of GLI3 to maintain Hh signaling in an inactive state.

Hedgehog signaling is essential for vertebrate development, however, less is known about the negative regulators that influence this pathway during the differentiation of cell fates. Using the mouse P19 embryonal carcinoma cell model, Suppressor of Fused (SUFU), a negative regulator of the Hedgehog pathway, was investigated during retinoic acid-induced neural differentiation. We found Hedgehog signaling was activated in the early phase of neural differentiation but was inactive during terminal differentiation of neurons and astrocytes. This early activation was required for neural differentiation however, it alone was not sufficient to induce neural lineages. SUFU, which regulates signaling at the level of GLI, remained relatively unchanged during the differentiation process, but its loss through CRISPIR-Cas9 gene editing resulted in ectopic expression of Hedgehog target genes. Interestingly, these SUFU-deficient cells were unable to differentiate in the absence of retinoic acid, but when differentiated using retinoic acid, they showed delayed and decreased astrocyte differentiation; neuron differentiation was unaffected. Ectopic activation of Hh target genes in SUFU-deficient cells remained throughout retinoic acid-induced differentiation and this phenotype was accompanied by the loss of GLI3, despite the presence of the Gli3 message. Thus, the study indicates the proper timing and proportion of glial cell differentiation requires SUFU, and its normal regulation of GLI3 to maintain Hh signaling in an inactive state.

Pluripotent stem cells (PSCs) have been described in naïve or primed pluripotent states. Domestic dogs are useful translational models in regenerative medicine, but their embryonic stem cells (cESCs) remain narrowly investigated. Primed-like cESCs expanded in the presence of leukemia inhibitory factor and fibroblast growth factor 2 (LIF-FGF2) acquire features of naïve pluripotency when exposed to chemical inhibitors and LIF (2iL). However, proliferation of cESCs is influenced by the pluripotent state and is comparatively slower than human or mouse PSCs. We propose that different metabolic pathway activities support ATP generation and biomass accumulation necessary for LIF-FGF2 and 2iL cESC proliferation. We found that 2iL cESCs have greater respiratory capacity, altered mitochondrial chain complex stoichiometry and elevated mitochondrial polarization state. Yet, 2iL-enriched cESCs exhibited immature ultrastructure, including previously unrecognized changes to cristae organization. Enhanced ATP level in 2iL cESCs is associated with altered retrograde signalling, whereas LIF-FGF2 cESCs exhibit a lipogenic phenotype. Inhibition of oxidative phosphorylation impaired proliferation and ATP production in 2iL cESCs but not LIF-FGF2 cESCs, which remained sensitive to glycolysis inhibition. Our study reveals distinct bioenergetic mechanisms contributing to steady-state expansion of distinct canine pluripotent states that can be exploited to improve derivation and culture of canine PSCs.