Regenerative strategies for kidney engineering

The kidney is the most important organ for water homeostasis and waste excretion. It performs several important physiological functions for homeostasis: it filters the metabolic waste out of circulation, regulates body fluid balances, and acts as an immune regulator and modulator of cardiovascular physiology. The development of in vitro renal disease models with pluripotent stem cells (both human embryonic stem cells and induced pluripotent stem cells) and the generation of robust protocols for in vitro derivation of renal-specific-like cells from patient induced pluripotent stem cells have just emerged. Here we review major findings in the field of kidney regeneration with a major focus on the development of stepwise protocols for kidney cell production from human pluripotent stem cells and the latest advances in kidney bioengineering (i.e. decellularized kidney scaffolds and bioprinting). The possibility of generating renal-like three-dimensional structures to be recellularized with renal-derived induced pluripotent stem cells may offer new avenues to develop functional kidney grafts on-demand

Kidney organoids for disease modeling

The kidney is formed during development by reciprocal interactions between the ureteric bud (UB) and the metanephric mesenchyme (MM), which promote the induction of nephron patterning and differentiation. Traditionally, UB and MM cells including nephron progenitor cells (NPCs) have been very difficult to isolate and maintain in culture due to their propensity to differentiate when outside their developmental niche. Remarkably, in recent years researchers have succeeded in prolonging the lifespan of mouse [1], rat [1], and human [2] NPCs in vitro, offering an avenue to expand the current knowledge of mammalian kidney development, and eventually for disease modelling and drug screening studies. Alternatively, renal progenitors have also been generated from human pluripotent stem cells (hPSCs) by mimicking early kidney developmental signals in vitro. Recently, different laboratories have been able to partially reproduce kidney organogenesis in a dish using hPSCs, successfully generating so-called kidney organoids [3,4,5,6]. Kidney organoids contain self-organized nephron-like structures composed of early podocyte cell clusters connected to tubular structures expressing markers of proximal tubules, loops of Henle and distal tubules [3,4,5,6]. In addition, kidney organoids display proximal tubular functionality in vitro, showing selective endocytosis of dextran cargoes [5,6], as well as responding to nephrotoxic agents [4,5,6]

Genome editing in human pluripotent stem cells: a systematic approach unrevealing pancreas development and disease

Although mouse models have represented a major tool for understanding and predicting molecular mechanisms responsible for several human genetic diseases, still species-specific differences between mouse and humans in their biochemical and physiological characteristics represent a major hurdle when translating promising findings into the human setting (1). For instance, in several types of maturity onset diabetes of the young (MODY; autosomal dominant), mice with heterozygous mutations do not develop diabetes (2). In this regard, the derivation of human embryonic stem cells (hESCs) in 1998 represented an unprecedented opportunity for human disease modelling, and a promising source for cell replacement therapies (3). Later on, the possibility to generate patient-derived induced pluripotent stem cells (iPSCs) has opened new venues for the potential translation of stem-cell related studies into the clinic (4)

Cèl·lules mare embrionàries i medicina regenerativa

Embryonary stem cells and Regenerative Medicine.Embryonary stem cells have represented a scientific revolution in recent years given their potential capacity to turn into any cell type in the organism. Their therapeutic importance lies in the fact that they can be useful for treating diseases caused by a deficit in cell functioning. Numerous research groups world-wide are working to find out the differentiation mechanisms, as well as alternative techniques to using preembryos, in order to discover a safe and efficient therapy for these types of illness

Regeneration and reprogramming compared

<p>Abstract</p> <p>Background</p> <p>Dedifferentiation occurs naturally in mature cell types during epimorphic regeneration in fish and some amphibians. Dedifferentiation also occurs in the induction of pluripotent stem cells when a set of transcription factors (<it>Oct4, Sox2, Klf4 </it>and <it>c-Myc</it>) is over expressed in mature cell types.</p> <p>Results</p> <p>We hypothesised that there are parallels between dedifferentiation or reprogramming of somatic cells to induced pluripotent stem cells and the natural process of dedifferentiation during epimorphic regeneration. We analysed expression levels of the most commonly used pluripotency associated factors in regenerating and non-regenerating tissue and compared them with levels in a pluripotent reference cell. We found that some of the pluripotency associated factors (<it>oct4/pou5f1, sox2, c-myc, klf4, tert, sall4, zic3, dppa2/4 </it>and <it>fut1</it>, a homologue of <it>ssea1</it>) were expressed before and during regeneration and that at least two of these factors (<it>oct4, sox2</it>) were also required for normal fin regeneration in the zebrafish. However these factors were not upregulated during regeneration as would be expected if blastema cells acquired pluripotency.</p> <p>Conclusions</p> <p>By comparing cells from the regeneration blastema with embryonic pluripotent reference cells we found that induced pluripotent stem and blastema cells do not share pluripotency. However, during blastema formation some of the key reprogramming factors are both expressed and are also required for regeneration to take place. We therefore propose a link between partially reprogrammed induced pluripotent stem cells and the half way state of blastema cells and suggest that a common mechanism might be regulating these two processes.</p

Skeletal muscle regeneration in Xenopus tadpoles and zebrafish larvae

<p>Abstract</p> <p>Background</p> <p>Mammals are not able to restore lost appendages, while many amphibians are. One important question about epimorphic regeneration is related to the origin of the new tissues and whether they come from mature cells via dedifferentiation and/or from stem cells. Several studies in urodele amphibians (salamanders) indicate that, after limb or tail amputation, the multinucleated muscle fibres do dedifferentiate by fragmentation and proliferation, thereby contributing to the regenerate. In <it>Xenopus laevis </it>tadpoles, however, it was shown that muscle fibres do not contribute directly to the tail regenerate. We set out to study whether dedifferentiation was present during muscle regeneration of the tadpole limb and zebrafish larval tail, mainly by cell tracing and histological observations.</p> <p>Results</p> <p>Cell tracing and histological observations indicate that zebrafish tail muscle do not dedifferentiate during regeneration. Technical limitations did not allow us to trace tadpole limb cells, nevertheless we observed no signs of dedifferentiation histologically. However, ultrastructural and gene expression analysis of regenerating muscle in tadpole tail revealed an unexpected dedifferentiation phenotype. Further histological studies showed that dedifferentiating tail fibres did not enter the cell cycle and <it>in vivo </it>cell tracing revealed no evidences of muscle fibre fragmentation. In addition, our results indicate that this incomplete dedifferentiation was initiated by the retraction of muscle fibres.</p> <p>Conclusions</p> <p>Our results show that complete skeletal muscle dedifferentiation is less common than expected in lower vertebrates. In addition, the discovery of incomplete dedifferentiation in muscle fibres of the tadpole tail stresses the importance of coupling histological studies with <it>in vivo </it>cell tracing experiments to better understand the regenerative mechanisms.</p

Characterization of the Epigenetic Changes During Human Gonadal Primordial Germ Cells Reprogramming

Epigenetic reprogramming is a central process during mammalian germline development. Genome-wide DNA demethylation in primordial germ cells (PGCs) is a prerequisite for the erasure of epigenetic memory, preventing the transmission of epimutations to the next generation. Apart from DNA demethylation, germline reprogramming has been shown to entail reprogramming of histone marks and chromatin remodelling. Contrary to other animal models, there is limited information about the epigenetic dynamics during early germ cell development in humans. Here, we provide further characterization of the epigenetic configuration of the early human gonadal PGCs. We show that early gonadal human PGCs are DNA hypomethylated and their chromatin is characterized by low H3K9me2 and high H3K27me3 marks. Similarly to previous observations in mice, human gonadal PGCs undergo dynamic chromatin changes concomitant with the erasure of genomic imprints. Interestingly, and contrary to mouse early germ cells, expression of BLIMP1/PRDM1 persists in through all gestational stages in human gonadal PGCs and is associated with nuclear lysine-specific demethylase-1. Our work provides important additional information regarding the chromatin changes associated with human PGCs development between 6 and 13 weeks of gestation in male and female gonads

Theoretical and experimental approaches to understand morphogen gradients

Morphogen gradients, which specify different fates for cells in a direct concentration-dependent manner, are a highly influential framework in which pattern formation processes in developmental biology can be characterized. A common analysis approach is combining experimental and theoretical strategies, thereby fostering relevant data on the dynamics and transduction of gradients. The mechanisms of morphogen transport and conversion from graded information to binary responses are some of the topics on which these combined strategies have shed light. Herein, we review these data, emphasizing, on the one hand, how theoretical approaches have been helpful and, on the other hand, how these have been combined with experimental strategies. In addition, we discuss those cases in which gradient formation and gradient interpretation at the molecular and/or cellular level may influence each other within a mutual feedback loop. To understand this interplay and the features it yields, it becomes essential to take system-level approaches that combine experimental and theoretical strategies

Tbx2 and Tbx3 Regulate the Dynamics of Cell Proliferation during Heart Remodeling

BACKGROUND: The heart forms from a linear tube that is subject to complex remodeling during embryonic development. Hallmarks of this remodeling are the looping of the heart tube and the regionalization into chamber and non-chamber myocardium. Cardiomyocytes in the future chamber myocardium acquire different cellular and physiological characteristics through activation of a chamber-specific genetic program, which is in part mediated by T-box genes. METHODOLOGY/PRINCIPAL FINDING: We characterize two new zebrafish T-box transcription factors, tbx3b and tbx2a, and analyze their role during the development of the atrioventricular canal. Loss- and gain-of-function analyses demonstrate that tbx3b and tbx2a are necessary to repress the chamber-genetic program in the non-chamber myocardium. We also show that tbx3b and tbx2a are required to control cell proliferation in the atrioventricular canal and that misregulation of cell proliferation in the heart tube influences looping. Furthermore, we characterize the heart phenotype of a novel Tbx3 mutation in mice and show that both the control of cell proliferation and the repression of chamber-specific genetic program in the non-chamber myocardium are conserved roles of Tbx3 in this species. CONCLUSIONS/SIGNIFICANCE: Taken together, our results uncover an evolutionarily conserved role of Tbx2/3 transcription factors during remodeling of the heart myocardium and highlight the importance of controlling cell proliferation as a driving force of morphogenesis

c‑MYC Triggers Lipid Remodelling During Early Somatic Cell Reprogramming to Pluripotency

Metabolic rewiring and mitochondrial dynamics remodelling are hallmarks of cell reprogramming, but the roles of the reprogramming factors in these changes are not fully understood. Here we show that c-MYC induces biosynthesis of fatty acids and increases the rate of pentose phosphate pathway. Time-course profiling of fatty acids and complex lipids during cell reprogramming using lipidomics revealed a profound remodelling of the lipid content, as well as the saturation and length of their acyl chains, in a c-MYC-dependent manner. Pluripotent cells displayed abundant cardiolipins and scarce phosphatidylcholines, with a prevalence of monounsaturated acyl chains. Cells undergoing cell reprogramming showed an increase in mitochondrial membrane potential that paralleled that of mitochondrial-specific cardiolipins. We conclude that c-MYC controls the rewiring of somatic cell metabolism early in cell reprogramming by orchestrating cell proliferation, synthesis of macromolecular components and lipid remodelling, all necessary processes for a successful phenotypic transition to pluripotency