Low Proliferative Capacity Research Articles

The chromatin landscape of high-grade serous ovarian cancer metastasis identifies regulatory drivers in post-chemotherapy residual tumour cells

Disease recurrence following chemotherapy is a major clinical challenge in ovarian cancer (OC), but little is known regarding how the tumour epigenome regulates transcriptional programs underpinning chemoresistance. We determine the single cell chromatin accessibility landscape of omental OC metastasis from treatment-naïve and neoadjuvant chemotherapy-treated patients and define the chromatin accessibility profiles of epithelial, fibroblast, myeloid and lymphoid cells. Epithelial tumour cells display open chromatin regions enriched with motifs for the oncogenic transcription factors MEIS and PBX. Post chemotherapy microenvironments show profound tumour heterogeneity and selection for cells with accessible chromatin enriched for TP53, TP63, TWIST1 and resistance-pathway-activating transcription factor binding motifs. An OC chemoresistant tumour subpopulation known to be present prior to treatment, and characterised by stress-associated gene expression, is enriched post chemotherapy. Nuclear receptors RORa, NR2F6 and HNF4G are uncovered as candidate transcriptional drivers of these cells whilst closure of binding sites for E2F2 and E2F4 indicate post-treated tumour having low proliferative capacity. Delineation of the gene regulatory landscape of ovarian cancer cells surviving chemotherapy treatment therefore reveals potential core transcriptional regulators of chemoresistance, suggesting novel therapeutic targets for improving clinical outcome.

Mechanically stimulated osteocytes maintain tumor dormancy in bone metastasis of non-small cell lung cancer by releasing small extracellular vesicles

Although preclinical and clinical studies have shown that exercise can inhibit bone metastasis progression, the mechanism remains poorly understood. Here, we found that non-small cell lung cancer (NSCLC) cells adjacent to bone tissue had a much lower proliferative capacity than the surrounding tumor cells in patients and mice. Subsequently, it was demonstrated that osteocytes, sensing mechanical stimulation generated by exercise, inhibit NSCLC cell proliferation and sustain the dormancy thereof by releasing small extracellular vesicles with tumor suppressor micro-RNAs, such as miR-99b-3p. Furthermore, we evaluated the effects of mechanical loading and treadmill exercise on the bone metastasis progression of NSCLC in mice. As expected, mechanical loading of the tibia inhibited the bone metastasis progression of NSCLC. Notably, bone metastasis progression of NSCLC was inhibited by moderate exercise, and combinations with zoledronic acid had additive effects. Moreover, exercise preconditioning effectively suppressed bone metastasis progression. This study significantly advances the understanding of the mechanism underlying exercise-afforded protection against bone metastasis progression.

Mechanically stimulated osteocytes maintain tumor dormancy in bone metastasis of non-small cell lung cancer by releasing small extracellular vesicles.

Although preclinical and clinical studies have shown that exercise can inhibit bone metastasis progression, the mechanism remains poorly understood. Here, we found that non-small cell lung cancer (NSCLC) cells adjacent to bone tissue had a much lower proliferative capacity than the surrounding tumor cells in patients and mice. Subsequently, it was demonstrated that osteocytes, sensing mechanical stimulation generated by exercise, inhibit NSCLC cell proliferation and sustain the dormancy thereof by releasing small extracellular vesicles with tumor suppressor micro-RNAs, such as miR-99b-3p. Furthermore, we evaluated the effects of mechanical loading and treadmill exercise on the bone metastasis progression of NSCLC in mice. As expected, mechanical loading of the tibia inhibited the bone metastasis progression of NSCLC. Notably, bone metastasis progression of NSCLC was inhibited by moderate exercise, and combinations with zoledronic acid had additive effects. Moreover, exercise preconditioning effectively suppressed bone metastasis progression. This study significantly advances the understanding of the mechanism underlying exercise-afforded protection against bone metastasis progression.

Magnetotactic hepatocytes promote liver repopulation after transplantation

Clinical application of hepatocyte transplantation has been halted due to the low engraftment efficiency and limited proliferation capacity of donor hepatocytes in the host liver. Here, we developed a magneto-plasmonic nanohybrid to spatiotemporally drive the primary hepatocytes into the host liver under the external magnetic field that significantly improves the engraftment efficiency of donor hepatocytes. A magnetic lipid-coated-gold-nanorods/iron-oxide nanocluster (GNR-IO) (LGI) was designed for efficient magneto-actuation and photoacoustic imaging-guided cell tracking during the hepatocyte transplantation. By optimizing the concentration of LGI (100 μg /mL) and the configuration of a magnetic field (260 mT), we were able to increase the engraftment efficiency of primary hepatocytes into the host livers of Fah-/- mice, a model with the genetic deficit of fumarylacetoacetate hydrolase (FAH) resulting in liver injuries, for 3.89-fold of the control by one week of the transplantation. Further assessment of repopulation of hepatocytes in the host liver indicated the LGI (100 μg /mL) + magnetic field (260 mT) group had up to 15 % repopulation of the host liver by three-weeks-post-transplantation and reached full repopulation by 6-weeks-post-transplantation. This was 3 ∼ 5 times more rapid than the Fah-/- mice transplanted with hepatocytes without the LGI and the magnetic field. RNA-sequencing of the hepatocytes incubated with LGI indicated that the loading of LGIs enhanced the migration, survival, and engraftment of hepatocytes by regulating the tumor necrosis factor (TNF)-signaling pathway and reducing the macrophage in host liver. Furthermore, LGI-labeled hepatocytes could be tracked in host livers with photoacoustic imaging for at least one week post transplantation. Most of the LGI were cleared from the host systems via the kidneys by six weeks. The novel strategy established in this study, which utilized nanoparticles combined with the magnetic field to improve the efficiency of primary hepatocyte engraftment and repopulation, can provide a feasible scheme for the clinical application of hepatocyte transplantation in the treatment of liver failure.

Derivation and travelling wave analysis of phenotype-structured haptotaxis models of cancer invasion

AbstractWe formulate haptotaxis models of cancer invasion wherein the infiltrating cancer cells can occupy a spectrum of states in phenotype space, ranging from ‘fully mesenchymal’ to ‘fully epithelial’. The more mesenchymal cells are those that display stronger haptotaxis responses and have greater capacity to modify the extracellular matrix (ECM) through enhanced secretion of matrix-degrading enzymes (MDEs). However, as a trade-off, they have lower proliferative capacity than the more epithelial cells. The framework is multiscale in that we start with an individual-based model that tracks the dynamics of single cells, which is based on a branching random walk over a lattice representing both physical and phenotype space. We formally derive the corresponding continuum model, which takes the form of a coupled system comprising a partial integro-differential equation for the local cell population density function, a partial differential equation for the MDE concentration and an infinite-dimensional ordinary differential equation for the ECM density. Despite the intricacy of the model, we show, through formal asymptotic techniques, that for certain parameter regimes it is possible to carry out a detailed travelling wave analysis and obtain invading fronts with spatial structuring of phenotypes. Precisely, the most mesenchymal cells dominate the leading edge of the invasion wave and the most epithelial (and most proliferative) dominate the rear, representing a bulk tumour population. As such, the model recapitulates similar observations into a front to back structuring of invasion waves into leader-type and follower-type cells, witnessed in an increasing number of experimental studies over recent years.

Efficient Generation of CRISPR/Cas9-Mediated Knockout Human Primary Keratinocytes by Electroporation.

Due to their full differentiation capacity in vitro, the culture of human primary keratinocytes (HPKs) represents a physiological model for answering basic biological and dermatological research questions, including those related to skin diseases and the investigation of treatment options. When modified with the CRISPR/Cas9 gene editing approach and cultivated in organotypic 3D epidermal equivalents (EEs), these human cells have the potential to replace established mouse models. However, even when cultivated on feeder cells, HPKs have only a low proliferation capacity in 2D culture, limiting their application potential. This is particularly true for CRISPR/Cas9-modified HPKs, whose generation commonly requires selection of targeted cells, negatively affecting their lifespan. Here, we describe a robust protocol for the rapid, simple, and efficient generation of single- and multi-gene CRISPR/Cas9 knockout HPKs by electroporation of ribonucleoprotein (RNP) complexes, which comprise one or multiple guide RNAs (gRNAs) and Cas9 protein. Unlike DNA transfection or virus-based targeting strategies, electroporation of RNPs represents a targeting approach that minimizes immunological and toxic side effects. Using efficient gRNAs results in the generation of HPKs with a high yield of knockout cells, allowing for their immediate use in experiments without requiring the laborious process of selecting targeted cells or maintaining a feeder cell culture. Furthermore, the use of RNPs and their delivery via electroporation minimizes off-target and other unspecific effects, preventing unintended genomic alterations. Most importantly, CRISPR/Cas9 knockout HPKs generated with this protocol have the ability to form a fully differentiated epidermis in 3D, thus facilitating the understanding of specific protein functions in a highly physiological human skin model. Alternatively, this approach proves valuable for generating models of mono- or polygenic skin diseases via knockouts, providing insights into the underlying molecular mechanisms and facilitating the development of novel therapeutic approaches.

Optogenetic Stimulation of the Cardiac Vagus Nerve to Promote Heart Regenerative Repair after Myocardial Infarction.

Background: It had been shown that selective cardiac vagal activation holds great potential for heart regeneration. Optogenetics has clinical translation potential as a novel means of modulating targeted neurons. This study aimed to investigate whether cardiac vagal activation via optogenetics could improve heart regenerative repair after myocardial infarction (MI) and to identify the underlying mechanism. Methods: We used an adeno-associated virus (AAV) as the vector to deliver ChR2, a light-sensitive protein, to the left nodose ganglion (LNG). To assess the effects of the cardiac vagus nerve on cardiomyocyte (CM) proliferation and myocardial regeneration in vivo, the light-emitting diode illumination (470 nm) was applied for optogenetic stimulation to perform the gain-of-function experiment and the vagotomy was used as a loss-of-function assay. Finally, sequencing data and molecular biology experiments were analyzed to determine the possible mechanisms by which the cardiac vagus nerve affects myocardial regenerative repair after MI. Results: Absence of cardiac surface vagus nerve after MI was more common in adult hearts with low proliferative capacity, causing a poor prognosis. Gain- and loss-of-function experiments further demonstrated that optogenetic stimulation of the cardiac vagus nerve positively regulated cardiomyocyte (CM) proliferation and myocardial regeneration in vivo. More importantly, optogenetic stimulation attenuated ventricular remodeling and improved cardiac function after MI. Further analysis of sequencing results and flow cytometry revealed that cardiac vagal stimulation activated the IL-10/STAT3 pathway and promoted the polarization of cardiac macrophages to the M2 type, resulting in beneficial cardiac regenerative repair after MI. Conclusions: Targeting the cardiac vagus nerve by optogenetic stimulation induced macrophage M2 polarization by activating the IL-10/STAT3 signaling pathway, which obviously optimized the regenerative microenvironment and then improved cardiac function after MI.

Abstract 12887: Cyclin D2 Overexpressing and Hypoimmunogenic Cardiomyocyte Spheroid Transplantation Induced Endogenous Myocardial Regeneration in the Pig Heart After Myocardial Infarction

Background: hiPSCs derived cardiomyocytes (hiPSC-CMs) have very low proliferative capacity, while patient hiPSC-CMs may have disease phenotypes which compromise therapeutic efficacy and allogenic hiPSC-CMs are expected to induce an immune response during clinical use. In order to solve these problems, we established a hiPSC line by knocking-out two components of the human leukocyte antigen (HLA) and over-expressing myosin heavy chain driven cyclin D2 (CCND2) systems in hiPSCs ( HLA-KO/CCND2-OE hiPSCs). Aim: To determine the therapeutic efficacy and mechanism of the xeno-transplantation of HLA-KO/CCND2-OE hiPSCs derived cardiomyocytes ( HLA-KO/CCND2-OE hiPSC-CMs) in the pig hearts after myocardial ischemia/reperfusion (IR). Methods and Results: HLA-KO/CCND2-OE hiPSC-CM spheroids were differentiated in shaking flasks. Purified HLA-KO/CCND2-OE hiPSC-CM spheroids were intramyocardially injected into pig hearts after IR (the Spheroid Group), while control animals received basal medium injection (the IR Group). Cardiac magnetic resonance imaging (cMRI) showed that pig heart function improved significantly compared to the IR Group at 4 weeks after IR. This finding was associated with a dramatical reduction in the scar size as determined by late gadolinium enhancement measurement. Immunohistology showed that significantly increased porcine CMs expressed cell proliferation markers, such as Ki67, phospho-histone 3 (pH3), and symmetrical Aurora B, and abundant YAP protein expression in porcine CM nuclei in the porcine hearts of the Spheroid Group compared to the IR Group at 1 week after IR. Western Blot analysis of pig heart tissue showed significantly reduced expression of phospho-YAP in the Spheroid Group. snRNAseq showed that number of cycling porcine CMs was > 5 folds in the Spheroid Groups than that in the IR Group. Angiogenic microarray showed that HLA-KO/CCND2-OE hiPSC-CM spheroids produced abundant follistatin which induced YAP protein expression and cytokinesis in CMs. Conclusions: Transplantation of HLA-KO/CCND2-OE hiPSC-CM spheroids effectively recovered porcine heart function, reduced scar size, and induced porcine CM proliferation through modulating YAP protein expression by follistatin secreted by transplanted cells.

Abstract 12975: Metabolic Reprogramming in Adult Cardiomyocytes Promotes Heart Regeneration

Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes. However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. We tested if dedifferentiating or reprogramming adult cardiomyocytes may recover their proliferative capability and thus promote heart regeneration after injury in mice.We bred mice with inducible, cardiac specific expression of the Yamanaka OSKM factors, enabling adult cardiomyocyte reprogramming and dedifferentiation in vivo. Two days after OSKM induction, adult cardiomyocytes presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult cardiomyocytes and recapitulated cardiomyocyte dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to cardiomyocyte injury, thus allowing cell dedifferentiation and proliferation as a regenerative response. (Cheng et al . Circulation 2022).

Heterogeneous Characteristics of the CD90+ Progenitors in the Fibrocartilage of Different Joints.

This study aimed to isolate and compare the mesenchymal stem cell characteristics of CD90+ cells from different fibrocartilage tissues in the temporomandibular joint (TMJ), the knee joint, and the intervertebral joint to further understand the similarities and differences of these 4 fibrocartilage tissues. CD90+ cells were isolated from TMJ disc, condylar cartilage, meniscus, and intervertebral disc by using magnetic-activated cell sorting. Cellular assays including 4.5-ethynyl-2'-deoxyuridine labeling, multilineage differentiation, colony formation, and cell migration were conducted to compare their mesenchymal stem cell characteristics. Immunofluorescent staining was performed for observing the expression of actively proliferating CD90+ cells within the tissues. H&E staining and Safranine O staining were used to compare the histological features. The CD90+ cells derived from these 4 fibrocartilage tissues exhibited comparable cell proliferation abilities. However, the cells from the TMJ disc displayed limited multilineage differentiation potential, colony formation, and cell migration abilities in comparison with the cells from the other fibrocartilage tissues. In vivo, there was relatively more abundant expression of CD90+ cells in the TMJ disc during the early postnatal stage. The limited EDU+ cell numbers signified a low proliferation capacity of CD90+ cells in the TMJ disc. In addition, we observed a significant decrease in cell density and a restriction in the synthesis of extracellular proteoglycans in the TMJ disc. Our study highlights the spatial heterogeneity of CD90+ cells in the fibrocartilages of different joint tissues, which may contribute to the limited cartilage repair capacity in the TMJ disc.

Downregulation of Swine Leukocyte Antigen Expression Decreases the Strength of Xenogeneic Immune Responses towards Renal Proximal Tubular Epithelial Cells.

Xenotransplantation reemerged as a promising alternative to conventional transplantation enlarging the available organ pool. However, success of xenotransplantation depends on the design and selection of specific genetic modifications and on the development of robust assays allowing for a precise assessment of tissue-specific immune responses. Nevertheless, cell-based assays are often compromised by low proliferative capacity of primary cells. Proximal tubular epithelial cells (PTECs) play a crucial role in kidney function. Here, we generated immortalized PTECs (imPTECs) by overexpression of simian virus 40 T large antigen. ImPTECs not only showed typical morphology and phenotype, but, in contrast to primary PTECs, they maintained steady cell cycling rates and functionality. Furthermore, swine leukocyte antigen (SLA) class I and class II transcript levels were reduced by up to 85% after transduction with lentiviral vectors encoding for short hairpin RNAs targeting β2-microglobulin and the class II transactivator. This contributed to reducing xenogeneic T-cell cytotoxicity (p < 0.01) and decreasing secretion of pro-inflammatory cytokines such as IL-6 and IFN-γ. This study showed the feasibility of generating highly proliferative PTECs and the development of tissue-specific immunomonitoring assays. Silencing SLA expression on PTECs was demonstrated to be an effective strategy to prevent xenogeneic cellular immune responses and may strongly support graft survival after xenotransplantation.

Abstract P1108: Development Of A Vascularized Cardiac Patch Using 3D Bioprinting To Regenerate Injured Myocardium

Introduction: Regeneration of the myocardium after ischemic injuries has remained a challenge due to the low proliferative capacity of cardiomyocytes (CMs). With the advance of tissue engineering, various cardiac patch devices have been developed and shown to improve cardiac tissue structure/function after myocardial infarction (MI). However, the therapeutic outcomes of patch devices have been limited by the lack of biological and structural complexity. Leveraging 3D bioprinting and stem cell technologies, this study presents the development of a next generation cardiac patch, integrated with heterogenous multi-cellular architecture, perfusable vasculature, and pro-proliferative molecules. Methods: A hybrid functional bioink, consisting of gelatin methacrylate (GelMA), gelatin, and fibrin, was optimized to fabricate the patch constructs. Two small molecule-based CM expansion techniques were employed, utilizing follistatin-like protein 1 (FSTL1) and CHIR99021 (CHIR), to further enhance the regenerative capacity of the patch. Complex vascular geometries were created by bioprinting sacrificial pluronic bioink in between two cellular composite bioink layers, containing human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and endothelial cells (ECs). The sacrificial ink was subsequently removed to create vascular channels, which were then endothelialized with ECs. Results: Bioprinted patches were successfully cultured under static and dynamic (physiologic flow) culture in vitro . The hiPSC-CMs encapsulated in gelMA-gelatin-fibrin bioink showed adequate long-term viability and function within the 3D environment. The printed vascular channels formed rather uniform endothelium. Both the FSTL1 and CHIR treatments resulted in significant in-situ expansion of hiPSC-CMs within bioprinted cardiac constructs. Further, testing the engineered patch in a rat MI model demonstrated the significant cardioprotective effect of patch. Conclusion: Together, this study introduces a new generation of bioprinted myocardial tissue analogues as a robust research-enabling platform for in vitro testing, and as a novel patch device with markedly enhanced regenerative capacity to treat diseased/damaged adult mammalian heart.

Combinational Overexpression of &lt;i&gt;Foxa3&lt;/i&gt; and &lt;i&gt;Hnf4a&lt;/i&gt; Enhance the Proliferation and Prolong the Functional Maintenance of Primary Hepatocytes

In an in vitro culture system, primary hepatocytes usually display a low proliferation capacity, accompanied with a decrease of viability and a loss of hepatocyte-specific functions. Previous studies have demonstrated that the combination introductions of certain hepatocyte-specific transcription factors are able to convert fibroblasts into functional hepatocyte-like cells. However, such combinational usage of transcription factors in primary hepatocytes culture has not yet sufficiently studied. The forkhead box protein A3 (FoxA3) and hepatocyte nuclear factor 4α (Hnf4α) are liver-enriched transcription factors that play vital roles in the differentiation, and maintenance of hepatocytes. Thus, we simultaneously overexpressed the two genes, Foxa3 and Hnf4a, in rat hepatocytes and observed that the combinational augmentation of these two transcription factors have enhanced the proliferation and stabilized the hepatocyte-specific functions of primary hepatocytes over a long-term culture period.

Development of a method of passaging and freezing human iPS cell-derived hepatocytes to improve their functions.

Human induced pluripotent stem (iPS) cell-derived hepatocyte-like cells (HLCs) are expected to replace primary human hepatocytes as a new source of functional hepatocytes in various medical applications. However, the hepatic functions of HLCs are still low and it takes a long time to differentiate them from human iPS cells. Furthermore, HLCs have very low proliferative capacity and are difficult to be passaged due to loss of hepatic functions after reseeding. To overcome these problems, we attempted to develop a technology to dissociate, cryopreserve, and reseed HLCs in this study. By adding epithelial-mesenchymal transition inhibitors and optimizing the cell dissociation time, we have developed a method for passaging HLCs without loss of their functions. After passage, HLCs showed a hepatocyte-like polygonal cell morphology and expressed major hepatocyte marker proteins such as albumin and cytochrome P450 3A4 (CYP3A4). In addition, the HLCs had low-density lipoprotein uptake and glycogen storage capacity. The HLCs also showed higher CYP3A4 activity and increased gene expression levels of major hepatocyte markers after passage compared to before passage. Finally, they maintained their functions even after their cryopreservation and re-culture. By applying this technology, it will be possible to provide ready-to-use availability of cryopreserved HLCs for drug discovery research.

Ectopic Purkinje fibers recruitment leads to permanent structural and functional conductive defects following myocardial infarction in newborn mice

The Purkinje network (PN) is the most distal part of the cardiac conduction system and ensures fast conduction of the electrical impulse through the ventricles. Despite its small volume in the heart, the PN has a disproportionally high pathogenic role. Indeed, defects in PN architecture or electrophysiology trigger conduction blocks and/or ventricular arrythmia associated with poor clinical outcomes. Purkinje fibers (PF) are specialized cardiomyocytes with a low proliferative capacity and are recruited from bipotent cardiomyocyte progenitors throughout development up to the neonatal period. Late PF recruitment is dependent on maximal levels of the transcription factor Nkx2-5. A decade ago, a neonatal regenerative window was identified in mice following myocardial infarction (MI). While damaged contractile myocardium regenerates within weeks, neither PN structure nor function has been investigated in this context. This study aims to determine whether the PN is capable of regeneration in the neonatal period, and whether PN maturation is compromised in the regenerating context. Neonatal MI was performed by permanent ligation of the left coronary artery on one-day-old mice. PN structure and function were characterized by histology and electrocardiogram. PF recruitment was investigated by clonal analysis in wildtype and Nkx2-5 haploinsufficent mice. Damage caused by the MI to the PN is spatially restricted and PFs are not replaced after regeneration, leaving a gap in the network. Surprisingly, we also observed PN hyperplasia (Fig. 1A) in regions that were not directly affected by the MI, attesting to abnormal PN maturation in the context of regeneration. At the cellular level, ectopic PFs have abnormal shapes and gap junction content. Consistently, ventricular conduction was slowed, shown by an increased QRS duration (Fig. 1B). Nkx2-5± mice, which are incompetent for late PF recruitment, do not develop MI-induced hyperplasia. Together with clonal analysis, this indicates that the hyperplastic PN in WT mice arises from ectopic PF recruitment during regeneration. Our study reveals the incapacity of the PN to regenerate after direct damage. We also uncover an extended window of PF recruitment following MI, which leads to permanent structural defects and impaired electrical conduction.

Combinational Overexpression of Foxa3 and Hnf4α Enhance the Proliferation and Prolong the Functional Maintenance of Primary Hepatocytes

In an in vitro culture system, primary hepatocytes usually display a low proliferation capacity, accompanied with a decrease of viability and a loss of hepatocyte-specific functions. Previous studies have demonstrated that the combination introductions of certain hepatocyte-specific transcription factors are able to convert fibroblasts into functional hepatocyte-like cells. However, such combinational usage of transcription factors in primary hepatocytes culture has not yet sufficiently studied. The forkhead box protein A3 (FoxA3) and hepatocyte nuclear factor 4α (Hnf4α) are liver-enriched transcription factors that play vital roles in the differentiation, and maintenance of hepatocytes. Thus, we simultaneously overexpressed the two genes, Foxa3 and Hnf4α, in rat hepatocytes and observed that the combinational augmentation of these two transcription factors have enhanced the proliferation and stabilized the hepatocyte-specific functions of primary hepatocytes over a long-term culture period.

Increased Perforin- and IL-21-Expressing NK Cells in Patients with Early-Stage Chronic Lymphocytic Leukemia

Objective: CD5+CD19+ cells have a low proliferation capacity and elevated expression of anti-apoptotic protein BCL-2, mostly in the G0/ G1 cell phase and accumulate in the pathogenesis of chronic lymphocytic leukemia (CLL). Natural killer (NK) cells have the ability to kill the intracellular pathogen-infected or cancer cells and secrete cytotoxic enzymes. This study evaluated the frequency, expression of cytotoxic enzymes, and intracellular cytokine levels of NK cells in CLL patients. Materials and Methods: In this study, peripheral blood mononuclear cells were isolated from peripheral blood samples of CLL patients (n=29) and healthy controls (n=16) by density gradient centrifugation method. The frequency of the total NK cells and the intracellular levels of perforin, granzyme, interferon (IFN)-γ, interleukin (IL)-21, IL-4 and IL-17 in NK cells were investigated. Results: Elevated total NK cell frequency were found in the CLL patients compared to the healthy controls, and negatively correlated with CD5+CD19+ malign B cell frequency. Increased perforin expression was observed in patients’ total NK cells. Additionally, increased levels of IL-17 and IL-21 in total NK cell of CLL patients were obtained compared to healthy subjects. Conclusion: The findings suggest that in the early stage of CLL, increased total NK cell frequency, and elevated perforin and IL-21 levels in NK cells might have a protective impact against the progression of the disease for CLL patients.

Metabolic Changes Associated With Cardiomyocyte Dedifferentiation Enable Adult Mammalian Cardiac Regeneration.

Cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, certain animals readily regenerate lost myocardium through a process involving dedifferentiation, which unlocks their proliferative capacities. We bred mice with inducible, CM-specific expression of the Yamanaka factors, enabling adult CM reprogramming and dedifferentiation in vivo. Two days after induction, adult CMs presented a dedifferentiated phenotype and increased proliferation in vivo. Microarray analysis revealed that upregulation of ketogenesis was central to this process. Adeno-associated virus-driven HMGCS2 overexpression induced ketogenesis in adult CMs and recapitulated CM dedifferentiation and proliferation observed during partial reprogramming. This same phenomenon was found to occur after myocardial infarction, specifically in the border zone tissue, and HMGCS2 knockout mice showed impaired cardiac function and response to injury. Finally, we showed that exogenous HMGCS2 rescues cardiac function after ischemic injury. Our data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to CM injury, thus allowing cell dedifferentiation and proliferation as a regenerative response.

Abstract 10234: HMGCS2-Mediated Ketogenesis Induces Adult Cardiomyocyte Dedifferentiation and Enables Cardiac Regeneration

Introduction: Human cardiac regeneration after injury is limited by the low proliferative capacity of adult mammalian cardiomyocytes (CMs). However, some animals readily regenerate lost myocardium via a process involving CM dedifferentiation, which unlocks their proliferative capacity. Methods: Inspired by this concept, we generated mice with inducible, CM-specific expression of the Yamanaka factors (OSKM), enabling adult CM dedifferentiation in vivo . We profiled changes in CM gene expression by microarray, measured metabolite production by LC-MS and 13 C NMR, and used adeno-associated virus (AAV) to over-express target genes. Cardiac function was measured by echocardiography and intraventricular catheterization. Results: Two days following OSKM induction, adult CMs exhibited a dedifferentiated phenotype and increased proliferation in vivo . Microarray analysis revealed alterations of metabolism-related pathways, particularly an increase of the ketogenic enzyme HMGCS2. Analysis of metabolites confirmed that HMGCS2-induced ketogenesis in adult CMs allowed a metabolic switch from fatty acid oxidation towards ketone utilization. AAV-driven overexpression of HMGCS2 in OSKM mice induced more adult CM dedifferentiation and increased the proliferation rate by two-fold compared to controls. The same phenomenon was observed after myocardial infarction, specifically in the border zone. On the contrary, CM-specific HMGCS2 knockout mice showed poorer baseline cardiac function and impaired recovery following injury. Forced expression of HMGCS2 rescued cardiac function in these mice, thus demonstrating a role of ketogenesis in CM dedifferentiation and proliferation. Conclusions: These data demonstrate the importance of HMGCS2-induced ketogenesis as a means to regulate metabolic response to mammalian cardiac injury, allowing cell dedifferentiation and proliferation as a regenerative response.

Drug-Containing Layered Double Hydroxide/Alginate Dispersions for Tissue Engineering

Alginate (Alg) is increasingly studied as a constitutive material of scaffolds for tissue engineering because of its easy gelation and biocompatibility, and the incorporation of drugs into its formulation allows for its functionality to be extended. However, Alg presents a low cell adhesion and proliferation capacity, and the incorporation of drugs may further reduce its biocompatibility. Layered double hydroxides (LDH) are promising fillers for Alg-based biomaterials, as they increase cell adhesion and interaction and provide drug storage and controlled release. In this work, LDH containing ibuprofen or naproxen were synthesized by coprecipitation at a constant pH and their properties upon their incorporation in Alg dispersions (LDH-Drug/Alg) were explored. Drug release profiles in simulated body fluid and the proliferation of pre-osteoblastic MC3T3-E1 cells by LDH-Drug/Alg dispersions were then evaluated, leading to results that confirm their potential as biomaterials for tissue engineering. They showed a controlled release with diffusive control, modulated by the in-situ formation of an Alg hydrogel in the presence of Ca2+ ions. Additionally, LDH-Drug/Alg dispersions mitigated the cytotoxic effects of the pure drugs, especially in the case of markedly cytotoxic drugs such as naproxen.