Co-emergence of cardiac and gut tissues promotes cardiomyocyte maturation within human iPSC-derived organoids.

During embryogenesis, paracrine signaling between tissues in close proximity contributes to the determination of their respective cell fate(s) and development into functional organs. Organoids are in vitro models that mimic organ formation and cellular heterogeneity, but lack the paracrine input of surrounding tissues. Here, we describe a human multilineage iPSC-derived organoid that recapitulates cooperative cardiac and gut development and maturation, with extensive cellular and structural complexity in both tissues. We demonstrate that the presence of endoderm tissue (gut/intestine) in the organoids contributed to the development of cardiac tissue features characteristic of stages after heart tube formation, including cardiomyocyte expansion, compartmentalization, enrichment of atrial/nodal cells, myocardial compaction, and fetal-like functional maturation. Overall, this study demonstrates the ability to generate and mature cooperative tissues originating from different germ lineages within a single organoid model, an advance that will further the examination of multi-tissue interactions during development, physiological maturation, and disease.

Retinal toxicity, in vivo and in vitro, associated with inhibition of nicotinamide phosphoribosyltransferase.

Nicotinamide phosphoribosyltransferase (NAMPT) is a pleiotropic protein with intra- and extra-cellular functions as an enzyme, cytokine, growth factor, and hormone. NAMPT is of interest for oncology, because it catalyzes the rate-limiting step in the salvage pathway to generate nicotinamide adenine dinucleotide (NAD), which is considered a universal energy- and signal-carrying molecule involved in cellular energy metabolism and many homeostatic functions. This manuscript describes NAMPT inhibitor-induced retinal toxicity that was identified in rodent safety studies. This toxicity had a rapid onset and progression and initially targeted the photoreceptor and outer nuclear layers. Using in vivo safety and efficacy rodent studies, human and mouse cell line potency data, human and rat retinal pigmented epithelial cell in vitro systems, and rat mRNA expression data of NAMPT, nicotinic acid phosphoribosyltransferase, and nicotinamide mononucleotide adenylyltransferease (NMNAT) in several tissues from rat including retina, we demonstrate that the retinal toxicity is on-target and likely human relevant. We demonstrate that this toxicity is not mitigated by coadministration of nicotinic acid (NA), which can enable NAD production through the NAMPT-independent pathway. Further, modifying the physiochemical properties of NAMPT inhibitors could not sufficiently reduce retinal exposure. Our work highlights opportunities to leverage appropriately designed efficacy studies to identify known and measurable safety findings to screen compounds more rapidly and reduce animal use. It also demonstrates that in vitro systems with the appropriate cell composition and relevant biology and toxicity endpoints can provide tools to investigate mechanism of toxicity and the human translation of nonclinical safety concerns.

Akt regulates vitamin D3-induced leukemia cell functional differentiation via Raf/MEK/ERK MAPK signaling.

1,25-dihydroxyvitamin D3 (vitamin D3) induces differentiation of HL-60 human myeloid leukemia cells; however, the signaling mechanism governing these effects is not fully clear. Here, we show that vitamin D3 induced functional differentiation by Akt through Raf/MEK/ERK MAPK signaling. Vitamin D3 downregulated Akt, weakened Akt-Raf1 interaction, and subsequently activated the Raf/MEK/ERK MAPK pathway. Pharmacological inhibition of MEK/ERK crippled differentiation in response to vitamin D3. Ectopic overexpression of Akt inhibited MAPK signaling, downregulated cyclin-dependent kinase (CDK) inhibitors p21(Wip1/Cip1) and p27(Kip1) and blunted differentiation in response to vitamin D3 while knockdown of Akt by RNA interference gave reverse effects. Furthermore, knockdown of the CDK inhibitors by siRNA crippled the recruitment of retinoblastoma protein (Rb) from the Raf1-Rb complex and Rb hypophosphorylation, and abolished differentiation in response to vitamin D3. Vitamin D3-induced MAPK signaling mediated upregulation of the CDK inhibitors and Rb, disassociation of Raf1 and Rb, and dephosphorylation of Rb, resulting in Rb binding to transcription factor E2F1 and subsequent differentiation. Finally, knockdown of Rb by siRNA prevented vitamin D3-induced differentiation. Mutating Rb at Ser795 evokes its association with E2F1, indicating the critical role of Rb Ser795 in regulating cell differentiation. Taken together, our data suggest that vitamin D3-triggered differentiation of human myeloid leukemia cells depends on downregulation of Akt, which dissociates from Raf1 and activates MAPK signaling leading to CDK inhibitor upregulation, Raf1 disassociation from Rb, and Rb upregulation and hypophosphorylation coupled to E2F1 binding.

Vitamin D3 induces autophagy of human myeloid leukemia cells.

Vitamin D3 causes potent suppression of various cancer cells; however, significant supraphysiological concentrations of this compound are required for antineoplastic effects. Current combinatorial therapies with vitamin D3 are restricted to differentiation effects. It remains uncertain if autophagy is involved in vitamin D3 inhibition on leukemia cells. Here we show that besides triggering differentiation and inhibiting apoptosis, which was previously known, vitamin D3 triggers autophagic death in human myeloid leukemia cells. Inhibiting differentiation does not efficiently diminish vitamin D3 suppression on leukemia cells. Vitamin D3 up-regulates Beclin1, which binds to class III phosphatidylinositol 3-kinase to trigger autophagy. Vitamin D3 phosphorylates Bad in its BH3 domain, resulting in disassociation of the apoptotic Bad-Bcl-xL complex and association of Bcl-xL with Beclin1 and ultimate suppression of apoptotic signaling. Knockdown of Beclin1 eliminates vitamin D3-induced autophagy and inhibits differentiation but activates apoptosis, suggesting that Beclin1 is required for both autophagy and differentiation, and autophagy cooperates with differentiation but excludes apoptosis, in which Beclin1 acts as an interface for these three different cascades. Moreover, additional up-regulation of autophagy, but not apoptosis, dramatically improves vitamin D3 inhibition on leukemia cells. These findings extend our understanding of the action of vitamin D3 in antineoplastic effects and the role of Beclin1 in regulating multiple cellular cascades and suggest a potentially promising strategy with a significantly better antileukemia effect.