Generation of mitochondria-rich kidney organoids from expandable intermediate mesoderm progenitors reprogrammed from human urine cells under defined medium.

BACKGROUND: The kidneys require vast amounts of mitochondria to provide ample energy to reabsorb nutrients and regulate electrolyte, fluid, and blood pressure homeostasis. The lack of the human model hinders the investigation of mitochondria homeostasis related to kidney physiology and disease. RESULTS: Here, we report the generation of mitochondria-rich kidney organoids via partial reprogramming of human urine cells (hUCs) under the defined medium. First, we reprogrammed mitochondria-rich hUCs into expandable intermediate mesoderm progenitor like cells (U-iIMPLCs), which in turn generated nephron progenitors and formed kidney organoids in both 2D and 3D cultures. Cell fate transitions were confirmed at each stage by marker expressions at the RNA and protein levels, along with chromatin accessibility dynamics. Single cell RNA-seq revealed hUCs-induced kidney organoids (U-iKOs) consist of podocytes, tubules, and mesenchyme cells with 2D dominated with mesenchyme and 3D with tubule and enriched specific mitochondria function associated genes. Specific cell types, such as podocytes and proximal tubules, loop of Henle, and distal tubules, were readily identified. Consistent with these cell types, 3D organoids exhibited the functional and structural features of the kidney, as indicated by dextran uptake and transmission electron microscopy. These organoids can be further matured in the chick chorioallantoic membrane. Finally, cisplatin, gentamicin, and forskolin treatment led to anatomical abnormalities typical of kidney injury and altered mitochondria homeostasis respectively. CONCLUSIONS: Our study demonstrates that U-iKOs recapitulate the structural and functional characteristics of the kidneys, providing a promising model to study mitochondria-related kidney physiology and disease in a personalized manner.

Regeneration of the human segmentation clock in somitoids in vitro.

Each vertebrate species appears to have a unique timing mechanism for forming somites along the vertebral column, and the process in human remains poorly understood at the molecular level due to technical and ethical limitations. Here, we report the reconstitution of human segmentation clock by direct reprogramming. We first reprogrammed human urine epithelial cells to a presomitic mesoderm (PSM) state capable of long-term self-renewal and formation of somitoids with an anterior-to-posterior axis. By inserting the RNA reporter Pepper into HES7 and MESP2 loci of these iPSM cells, we show that both transcripts oscillate in the resulting somitoids at ~5 h/cycle. GFP-tagged endogenous HES7 protein moves along the anterior-to-posterior axis during somitoid formation. The geo-sequencing analysis further confirmed anterior-to-posterior polarity and revealed the localized expression of WNT, BMP, FGF, and RA signaling molecules and HOXA-D family members. Our study demonstrates the direct reconstitution of human segmentation clock from somatic cells, which may allow future dissection of the mechanism and components of such a clock and aid regenerative medicine.

Appropriate Exogenous Expression Stoichiometry of GATA4 as an Important Factor for Cardiac Reprogramming of Human Dermal Fibroblasts.

Reprogramming of human dermal fibroblasts (HDFs) into induced cardiomyocyte-like cells (iCMs) represents a promising strategy for human cardiac regeneration. Different cocktails of cardiac transcription factors can convert HDFs into iCMs, although with low efficiency and immature phenotype. Here, GATA4, MEF2C, TBX5, MESP1, and MYOCD (GMTMeMy for short) were used to reprogram HDFs by retrovirus infection. We found that the exogenous expression stoichiometry of GATA4 (GATA4 stoichiometry) significantly affected reprogramming efficiency. When 1/8 dosage of GATA4 virus (GATA4 dosage) plus MTMeMy was used, the reprogramming efficiency was obviously improved compared with average pooled virus encoding each factor, which measured, by the expression level of cardiac genes, the percentage of cardiac troponin T and alpha-cardiac myosin heavy-chain immunopositive cells and the numbers of iCMs showing calcium oscillation or beating synchronously in co-culture with mouse CMs. In addition, we prepared conditioned maintenance medium (CMM) by CM differentiation of H9 human embryonic stem cell line. We found that compared with traditional maintenance medium (TMM), CMM made iCMs show well-organized sarcomere formation and characteristic calcium oscillation wave earlier. These findings demonstrated that appropriate GATA4 stoichiometry was essential for cardiac reprogramming and some components in CMM were important for maturation of iCMs.

MYOCD is Required for Cardiomyocyte-like Cells Induction from Human Urine Cells and Fibroblasts Through Remodeling Chromatin.

Despite direct reprogramming of human cardiac fibroblasts into induced cardiomyocytes (iCM) holds great potential for heart regeneration, the mechanisms are poorly understood. Whether other human somatic cells could be reprogrammed into cardiomyocytes is also unknown. Here, we report human urine cells (hUCs) could be converted into CM-like cells from different donors and the related chromatin accessibility dynamics (CAD) by assay for transposase accessible chromatin(ATAC)-seq. hUCs transduced by MEF2C, TBX5, MESP1 and MYOCD but without GATA4 expressed multiple cardiac specific genes, exhibited Ca2+ oscillation potential and sarcomeric structures, and contracted synchronously in coculture with mouse CM. Additionally, we found that MYOCD is required for both closing and opening critical loci, mainly by hindering the opening of loci enriched with motifs for the TEAD and AP1 family and promoting the closing of loci enriched with ETS motifs. These changes differ partially from CAD observed during iCM induction from human fibroblasts. Collectively, our study offers one practical platform for iCM generation and insights into mechanisms for iCM fate determination.

[Establishment of a prognostic Nomogram model for predicting the first 72-hour mortality in polytrauma patients].

OBJECTIVE: To establish a prognostic Nomogram model for predicting the risk of early death in polytrauma patients. METHODS: Data extracted from a polytrauma study on Dryad, an open access database, was selected for secondary analysis. Patients from 18 to 65 years old with polytrauma in the original data were included. All patients with missing variables, such as blood lactic acid (Lac), Glasgow coma score (GCS) and injury severity score (ISS) at admission, were excluded. The differences of gender, age, Lac, ISS and GCS scores between the patients who died within 72 hours and those who survived were analyzed. The risk factors for 72-hour death were analyzed by Logistic regression, and the Nomogram prediction model was established using R software. The receiver operating characteristic (ROC) curve was used to evaluate the predictive ability of the model, and the Bootstrap method was used for internal verification by repeating sample for 1 000 times. Decision curve (DCA) was applied to analyze the clinical practical value of the model. RESULTS: A total of 2 315 polytrauma patients were included. Logistic regression analysis showed that Lac, GCS score and age > 55 years old were the risk factors for early death in polytrauma patients [Lac: odds ratio (OR) = 1.36, 95% confidence interval (95%CI) was 1.29-1.42, P < 0.001; GCS score: OR = 0.76, 95%CI was 0.73-0.79, P < 0.001; age > 55 years old: OR = 1.92, 95%CI was 1.37-2.66, P < 0.001]. The prediction model was established by using the above risk factors and displayed by Nomogram. ROC curve analysis showed that the area under the ROC curve (AUC) of Nomogram model to predict the risk of death within 72 hours was 0.858, and the predictive ability of Nomogram model was significantly higher than that of Lac (AUC = 0.743), GCS score (AUC = 0.774) and ISS score (AUC = 0.699), all P < 0.05. The model calibration chart showed that the predicting probability was consistent with the actual occurrence probability, and the DCA showed that Nomogram model presented excellent clinical value in predicting the 72-hour death risk for polytrauma patients. CONCLUSIONS: The prognostic Nomogram model presents significantly predictive value for the risk of death within 72 hours in polytrauma patients. Prognostic Nomogram model could offer individualized, visualized and graphical prediction pattern, and provide physicians with practical diagnostic tool for triage system and management of polytrauma according to precision medicine.