The future of direct cardiac reprogramming: any GMT cocktail variety?

Direct cardiac reprogramming has emerged as a novel therapeutic approach to treat and regenerate injured hearts through the direct conversion of fibroblasts into cardiac cells. Most studies have focused on the reprogramming of fibroblasts into induced cardiomyocytes (iCMs). The first study in which this technology was described, showed that at least a combination of three transcription factors, GATA4, MEF2C and TBX5 (GMT cocktail), was required for the reprogramming into iCMs in vitro using mouse cells. However, this was later demonstrated to be insufficient for the reprogramming of human cells and additional factors were required. Thereafter, most studies have focused on implementing reprogramming efficiency and obtaining fully reprogrammed and functional iCMs, by the incorporation of other transcription factors, microRNAs or small molecules to the original GMT cocktail. In this respect, great advances have been made in recent years. However, there is still no consensus on which of these GMT-based varieties is best, and robust and highly reproducible protocols are still urgently required, especially in the case of human cells. On the other hand, apart from CMs, other cells such as endothelial and smooth muscle cells to form new blood vessels will be fundamental for the correct reconstruction of damaged cardiac tissue. With this aim, several studies have centered on the direct reprogramming of fibroblasts into induced cardiac progenitor cells (iCPCs) able to give rise to all myocardial cell lineages. Especially interesting are reports in which multipotent and highly expandable mouse iCPCs have been obtained, suggesting that clinically relevant amounts of these cells could be created. However, as of yet, this has not been achieved with human iCPCs, and exactly what stage of maturity is appropriate for a cell therapy product remains an open question. Nonetheless, the major concern in regenerative medicine is the poor retention, survival, and engraftment of transplanted cells in the cardiac tissue. To circumvent this issue, several cell pre-conditioning approaches are currently being explored. As an alternative to cell injection, in vivo reprogramming may face fewer barriers for its translation to the clinic. This approach has achieved better results in terms of efficiency and iCMs maturity in mouse models, indicating that the heart environment can favor this process. In this context, in recent years some studies have focused on the development of safer delivery systems such as Sendai virus, Adenovirus, chemical cocktails or nanoparticles. This article provides an in-depth review of the in vitro and in vivo cardiac reprograming technology used in mouse and human cells to obtain iCMs and iCPCs, and discusses what challenges still lie ahead and what hurdles are to be overcome before results from this field can be transferred to the clinical settings

Generation of two transgene-free human iPSC lines from CD133+ cord blood cells

We have generated two human induced pluripotent stem cell (iPSC) lines from CD133+ cells isolated from umbilical cord blood (CB) of a female child using non-integrative Sendai virus. Here we describe the complete characterization of these iPSC lines: PRYDi-CB5 and PRYDi-CB40

Generation of two transgene-free human iPSC lines from CD133+ cord blood cells

We have generated two human induced pluripotent stem cell (iPSC) lines from CD133+ cells isolated from umbilical cord blood (CB) of a female child using non-integrative Sendai virus. Here we describe the complete characterization of these iPSC lines: PRYDi-CB5 and PRYDi-CB40

Fast and efficient neural conversion of human hematopoietic cells

Neurons obtained directly from human somatic cells hold great promise for disease modeling and drug screening. Available protocols rely on overexpression of transcription factors using integrative vectors and are often slow, complex, and inefficient. We report a fast and efficient approach for generating induced neural cells (iNCs) directly from human hematopoietic cells using Sendai virus. Upon SOX2 and c-MYC expression, CD133-positive cord blood cells rapidly adopt a neuroepithelial morphology and exhibit high expansion capacity. Under defined neurogenic culture conditions, they express mature neuronal markers and fire spontaneous action potentials that can be modulated with neurotransmitters. SOX2 and c-MYC are also sufficient to convert peripheral blood mononuclear cells into iNCs. However, the conversion process is less efficient and resulting iNCs have limited expansion capacity and electrophysiological activity upon differentiation. Our study demonstrates rapid and efficient generation of iNCs from hematopoietic cells while underscoring the impact of target cells on conversion efficiency

Generation of NKX2.5(GFP) Reporter Human iPSCs and Differentiation Into Functional Cardiac Fibroblasts

Direct cardiac reprogramming has emerged as an interesting approach for the treatment and regeneration of damaged hearts through the direct conversion of fibroblasts into cardiomyocytes or cardiovascular progenitors. However, in studies with human cells, the lack of reporter fibroblasts has hindered the screening of factors and consequently, the development of robust direct cardiac reprogramming protocols.In this study, we have generated functional human NKX2.5(GFP) reporter cardiac fibroblasts. We first established a new NKX2.5(GFP) reporter human induced pluripotent stem cell (hiPSC) line using a CRISPR-Cas9-based knock-in approach in order to preserve function which could alter the biology of the cells. The reporter was found to faithfully track NKX2.5 expressing cells in differentiated NKX2.5(GFP) hiPSC and the potential of NKX2.5-GFP + cells to give rise to the expected cardiac lineages, including functional ventricular- and atrial-like cardiomyocytes, was demonstrated. Then NKX2.5(GFP) cardiac fibroblasts were obtained through directed differentiation, and these showed typical fibroblast-like morphology, a specific marker expression profile and, more importantly, functionality similar to patient-derived cardiac fibroblasts. The advantage of using this approach is that it offers an unlimited supply of cellular models for research in cardiac reprogramming, and since NKX2.5 is expressed not only in cardiomyocytes but also in cardiovascular precursors, the detection of both induced cell types would be possible. These reporter lines will be useful tools for human direct cardiac reprogramming research and progress in this field.This work was supported by PID 2019-107150RB-I00/AEI/ 10.13039/501100011033 to XC-V; by the “Ramón y Cajal” State Program, Ministry of Economy and Competitivenes

Regulation of Embryonic and Induced Pluripotency by Aurora Kinase-p53 Signaling

SummaryMany signals must be integrated to maintain self-renewal and pluripotency in embryonic stem cells (ESCs) and to enable induced pluripotent stem cell (iPSC) reprogramming. However, the exact molecular regulatory mechanisms remain elusive. To unravel the essential internal and external signals required for sustaining the ESC state, we conducted a short hairpin (sh) RNA screen of 104 ESC-associated phosphoregulators. Depletion of one such molecule, aurora kinase A (Aurka), resulted in compromised self-renewal and consequent differentiation. By integrating global gene expression and computational analyses, we discovered that loss of Aurka leads to upregulated p53 activity that triggers ESC differentiation. Specifically, Aurka regulates pluripotency through phosphorylation-mediated inhibition of p53-directed ectodermal and mesodermal gene expression. Phosphorylation of p53 not only impairs p53-induced ESC differentiation but also p53-mediated suppression of iPSC reprogramming. Our studies demonstrate an essential role for Aurka-p53 signaling in the regulation of self-renewal, differentiation, and somatic cell reprogramming

Generation of Functional Human NKX2.5GFP Reporter Cell Lines for Direct Reprogramming into Proliferative Cardiac Progenitors

The mammalian heart is the first organ formed in the embryo and is composed of four chambers: right atrium (RA), right ventricle (RV), left atrium (LA), and left ventricle (LV), and three layers: the endocardium, the myocardium, and the pericardium. The endocardium is formed by simple squamous epithelium known as endothelium, which lines the chambers and is joined to myocardium by a thin layer of connective tissue. The pericardium is comprised of two distinct sublayers: the inner serous pericardium, which is formed by visceral pericardium or epicardium and parietal pericardium separated by pericardial cavity, and the outer fibrous pericardium made of dense connective tissue. The myocardium is the muscular layer of the heart, it is the thickest one and is located between the endocardium and the pericardium. With regard to cellular composition, the heart is principally formed by cardiomyocytes (CM), cardiac fibroblasts (cFib) , vascular smooth muscle cells (localized fundamentally within the myocardium), vascular endothelial cells (located within the myocardium and endocardium) and mesothelial cells present in the pericardium 1–3 . CM are specialized cells with a complex filament structure responsible for the control of the rhythmic beating of the heart and present heterogeneity depending on the location, morphology, and function, including atrial, ventricular, sinoatrial nodal, atrioventricular nodal, His bundle, and Purkinje fibers 4,5

Identification of novel regulators of transcription in iPSC-derived cardiovascular progenitors

Stem cells allow to investigate about the basic mechanisms that regulate embryonic development, cellular plasticity, and organ maintenance and regeneration. Induced pluripotent stem cells (iPSCs) are a powerful source of cells for diverse applications such as developmental and disease modeling, drug discovery and regenerative medicine. Cardiac development is coordinated by complex interactions between cardiac progenitor cell populations, different molecular signaling pathways, and spatially and temporally regulated gene expression. Cardiovascular progenitors (CVPs), with similar potential to these present in early stages of embryonic development, can be obtained from iPSCs by mimicking signaling during cardiogenesis, creating an ideal cell source to treat the damaged heart. However, the mechanisms and conditions for long-term self-renewal and maintenance of CVPs remain elusive. The generation of new iPSC models for tracing CVP lineages permits to delve into the biology of CVPs and discover novel potential regulators of their fate. We have established three different Cre/LoxP mouse models for lineage tracing of CVPs and their cell progeny by the expression of ZsGreen (ZsG) protein: Ai6-Mesp1-Cre (Mesp1 tracer), Ai6-Isl1-Cre (Isl1 tracer) and Ai6-Mef2c-AHF-Cre (AHF tracer) mice. Multiple iPSC clones have been derived from Ai6-Isl1-Cre and Ai6-Mef2c-AHF-Cre reporter mice. Several generated iPSC lines have been fully characterized, demonstrating embryonic stem-like features. iPSCs encoded the expected genomic insertions, showed normal karyotypes, transgenes were silenced, and expressed endogenous pluripotency-associated markers. Moreover, iPSCs were capable to differentiate into the three germ layers both in vitro and in vivo. We have verified the utility of established AHFiPSCs to track CVPs and their differentiated progeny. Upon differentiation, ZsG+ cells derived from AHFiPSCs appeared from embryoid body (EB) day 6 onwards, expressed cardiovascular-related markers, and were able to differentiate into cardiomyocytes, endothelial and smooth muscle cells. Comparative gene expression analysis using four different AHFiPSC lines revealed distinct molecular signatures in three particular stages of differentiation: undifferentiated iPSCs (AHFiPS-D0), sorted ZsG+ cells at day 6 (AHFiPS-D6.ZsG+) and sorted ZsG+ cells at day 13 of differentiation (AHFiPS-D13.ZsG+), that expressed pluripotency-, CVP- and cardiac/vascular lineages-associated markers, respectively. Gapdh and Polr2a have been determined the most stable housekeeping genes along the differentiation of AHFiPSCs, being optimal for accurate normalization of gene expression in our samples. We have identified novel regulators of transcription specifically upregulated in CVPs: Lin28a (and its paralog Lin28b), Lhx1 and Nr6a1. The expression of these genes was also found increased in the corresponding CVP-enriched samples from two different public analyses using mouse and human pluripotent stem cells. Moreover, using bioinformatic analysis of biological pathways we have found p53 as an interconnecting molecule of all these selected regulators of transcription. In order to explore novel insights into the biological role of the selected regulators in CVP fate, we have generated an inducible vector (pTRE-CDS-IRES-Puro-REX1-Blast) to carry out gain-of-function (GOF) analyses. This Tet-On system worked properly in AHFiPSC clones, but unfortunately it failed to work in EBs of certain size along differentiation. In contrast, this GOF system correctly functioned in human iPSCs differentiated in monolayer cultures. We have established four CBiPS1sv-4F-5 cell lines carrying Tet-On systems for the inducible expression of LIN28A, LIN28B, NR6A1 and LHX1, and preliminary results indicated that these regulators of transcription might have a role in CVP fate determination

Mechanism of apoptosis induced by IFN-α in human myeloma cells: Role of Jak1 and Bim and potentiation by rapamycin

Interferon-α (IFN-α) has been used for the last 20 years in the maintenance therapy of multiple myeloma (MM), though it is only effective in some patients. Congruent with this, IFN-α induces apoptosis in some MM cell lines. Understanding the mechanism of IFN-α-induced apoptosis could be useful in establishing criteria of eligibility for therapy. Here we show that IFN-α-induced apoptosis in the MM cell lines U266 and H929 was completely blocked by a specific inhibitor of Jak1. The mTOR inhibitor rapamycin mitigated apoptosis in U266 but potentiated it in H929 cells. IFN-α induced PS exposure, ΔΨm loss and pro-apoptotic conformational changes of Bak, but not of Bax, and was fully prevented by Mcl-1 overexpression in U266 cells. IFN-α treatment caused the release of cytochrome c from mitochondria to cytosol and consequently, a limited proteolytic processing of caspases. Apoptosis induced by IFN-α was only slightly prevented by caspase inhibitors. Levels of the BH3-only proteins PUMA and Bim increased during IFN-α treatment. Bim increase and apoptosis was prevented by transfection with the siRNA for Bim. PUMA-siRNA transfection reduced electroporation-induced apoptosis but had no effect on apoptosis triggered by IFN-α. The potentiating effect of rapamycin on apoptosis in H929 cells was associated to an increase in basal and IFN-α-induced Bim levels. Our results indicate that IFN-α causes apoptosis in myeloma cells through a moderate triggering of the mitochondrial route initiated by Bim and that mTOR inhibitors may be useful in IFN-α maintenance therapy of certain MM patients.This work was supported by Myeloma Thematic Network grant G03/136 from Fondo de Investigaciones Sanitarias (Ministerio de Sanidad, Spain).Peer Reviewe

Generation of Functional Human NKX2.5GFP Reporter Cell Lines for Direct Reprogramming into Proliferative Cardiac Progenitors

The mammalian heart is the first organ formed in the embryo and is composed of four chambers: right atrium (RA), right ventricle (RV), left atrium (LA), and left ventricle (LV), and three layers: the endocardium, the myocardium, and the pericardium. The endocardium is formed by simple squamous epithelium known as endothelium, which lines the chambers and is joined to myocardium by a thin layer of connective tissue. The pericardium is comprised of two distinct sublayers: the inner serous pericardium, which is formed by visceral pericardium or epicardium and parietal pericardium separated by pericardial cavity, and the outer fibrous pericardium made of dense connective tissue. The myocardium is the muscular layer of the heart, it is the thickest one and is located between the endocardium and the pericardium. With regard to cellular composition, the heart is principally formed by cardiomyocytes (CM), cardiac fibroblasts (cFib) , vascular smooth muscle cells (localized fundamentally within the myocardium), vascular endothelial cells (located within the myocardium and endocardium) and mesothelial cells present in the pericardium 1–3 . CM are specialized cells with a complex filament structure responsible for the control of the rhythmic beating of the heart and present heterogeneity depending on the location, morphology, and function, including atrial, ventricular, sinoatrial nodal, atrioventricular nodal, His bundle, and Purkinje fibers 4,5