Carmen Escobedo-Lucea

Hypoxia Promotes Efficient Differentiation of Human Embryonic Stem Cells to Functional Endothelium

Early development of mammalian embryos occurs in an environment of relative hypoxia. Nevertheless, human embryonic stem cells (hESC), which are derived from the inner cell mass of blastocyst, are routinely cultured under the same atmospheric conditions (21% O2) as somatic cells. We hypothesized that O2 levels modulate gene expression and differentiation potential of hESC, and thus, we performed gene profiling of hESC maintained under normoxic or hypoxic (1% or 5% O2) conditions. Our analysis revealed that hypoxia downregulates expression of pluripotency markers in hESC but increases significantly the expression of genes associated with angio‐ and vasculogenesis including vascular endothelial growth factor and angiopoitein‐like proteins. Consequently, we were able to efficiently differentiate hESC to functional endothelial cells (EC) by varying O2 levels; after 24 hours at 5% O2, more than 50% of cells were CD34+. Transplantation of resulting endothelial‐like cells improved both systolic function and fractional shortening in a rodent model of myocardial infarction. Moreover, analysis of the infarcted zone revealed that transplanted EC reduced the area of fibrous scar tissue by 50%. Thus, use of hypoxic conditions to specify the endothelial lineage suggests a novel strategy for cellular therapies aimed at repair of damaged vasculature in pathologies such as cerebral ischemia and myocardial infarction. STEM CELLS 2010;28:407–418

Different gDNA content in the subpopulations of prostate cancer extracellular vesicles: Apoptotic bodies, microvesicles, and exosomes

Extracellular vesicles (EVs) are cell‐derived membrane vesicles. EVs contain several RNAs such as mRNA, microRNAs, and ncRNAs, but less is known of their genomic DNA (gDNA) content. It is also unknown whether the DNA cargo is randomly sorted or if it is systematically packed into specific EV subpopulations. The aim of this study was to analyze whether different prostate cancer (PCa) cell‐derived EV subpopulations (apoptotic bodies, microvesicles, and exosomes) carry different gDNA fragments.

Hippo pathway effectors control cardiac progenitor cell fate by acting as dynamic sensors of substrate mechanics and nanostructure

Stem cell responsiveness to extracellular matrix (ECM) composition and mechanical cues has been the subject of a number of investigations so far, yet the molecular mechanisms underlying stem cell mechano-biology still need full clarification. Here we demonstrate that the paralog proteins YAP and TAZ exert a crucial role in adult cardiac progenitor cell mechano-sensing and fate decision. Cardiac progenitors respond to dynamic modifications in substrate rigidity and nanopattern by promptly changing YAP/TAZ intracellular localization. We identify a novel activity of YAP and TAZ in the regulation of tubulogenesis in 3D environments and highlight a role for YAP/TAZ in cardiac progenitor proliferation and differentiation. Furthermore, we show that YAP/TAZ expression is triggered in the heart cells located at the infarct border zone. Our results suggest a fundamental role for the YAP/TAZ axis in the response of resident progenitor cells to the modifications in microenvironment nanostructure and mechanics, thereby contributing to the maintenance of myocardial homeostasis in the adult heart. These proteins are indicated as potential targets to control cardiac progenitor cell fate by materials design.

Nanofibrillar cellulose wound dressing in skin graft donor site treatment.

BACKGROUND Although new therapeutic approaches for burn treatment have made progress, there is still need for better methods to enhance wound healing and recovery especially in severely burned patients. Nanofibrillar cellulose (NFC) has gained attention due to its renewable nature, good biocompatibility and excellent physical properties that are of importance for a range of applications in pharmaceutical and biomedical fields. In the present study, we investigated the potential of a wood based NFC wound dressing in a clinical trial on burn patients. Previously, we have investigated NFC as a topical functionalized wound dressing that contributes to improve wound healing in mice. METHODS Wood based NFC wound dressing was tested in split-thickness skin graft donor site treatment for nine burn patients in clinical trials at Helsinki Burn Centre. NFC dressing was applied to split thickness skin graft donor sites. The dressing gradually dehydrated and attached to donor site during the first days. During the clinical trials, physical and mechanical properties of NFC wound dressing were optimized by changing its composition. From patient 5 forward, NFC dressing was compared to commercial lactocapromer dressing, Suprathel® (PMI Polymedics, Germany). RESULTS Epithelialization of the NFC dressing-covered donor site was faster in comparison to Suprathel®. Healthy epithelialized skin was revealed under the detached NFC dressing. NFC dressing self-detached after 11-21days for patients 1-9, while Suprathel® self-detached after 16-28days for patients 5-9. In comparison studies with patients 5-9, NFC dressing self-detached on average 4days earlier compared with Suprathel®. Lower NFC content in the material was evaluated to influence the enhanced pliability of the dressing and attachment to the wound bed. No allergic reaction or inflammatory response to NFC was observed. NFC dressing did not cause more pain for patients than the traditional methods to treat the skin graft donor sites. CONCLUSION Based on the preliminary clinical data, NFC dressing seems to be promising for skin graft donor site treatment since it is biocompatible, attaches easily to wound bed, and remains in place until donor site has renewed. It also detaches from the epithelialized skin by itself.

A Xenogeneic-Free Protocol for Isolation and Expansion of Human Adipose Stem Cells for Clinical Uses

Human adipose stem cells (hASCs) play a crucial role in the fields of regenerative medicine and tissue engineering for different reasons: the abundance of adipose tissue, their easy harvesting, the ability to multipotent differentiation and the fact that they do not trigger allogeneic blood response or secrete cytokines that act as immunosuppressants. The vast majority of protocols use animal origin reagents, with the underlying risk of transmitting infections by non-human pathogens. We have designed a protocol to isolate and maintain the properties of hASCs avoiding xenogeneic reagents. These changes not only preserve hASCs morphology, but also increase cell proliferation and maintain their stem cell marker profile. On the other hand, human serum albumin (HSA), Tryple® and human Serum (HS), do not affect hASCs multipotent differentiation ability. The amendments introduced do not trigger modifications in the transcriptional profile of hASCs, alterations in key biochemical pathways or malignization. Thus, we have proven that it is possible to isolate and maintain hASCs avoiding animal reagents and, at the same time, preserving crucial culture parameters during long term culture. Thereby we have revealed a novel and effective tool for the improvement of clinical, cell-based therapies.

DNA sequences within glioma-derived extracellular vesicles can cross the intact blood-brain barrier and be detected in peripheral blood of patients

Tumor-cell-secreted extracellular vesicles (EVs) can cross the disrupted blood-brain barrier (BBB) into the bloodstream. However, in certain gliomas, the BBB remains intact, which might limit EVs release. To evaluate the ability of tumor-derived EVs to cross the BBB, we used an orthotopic xenotransplant mouse model of human glioma-cancer stem cells featuring an intact BBB. We demonstrated that all types of tumor cells-derived EVs−apoptotic bodies, shedding microvesicles and exosomes−cross the intact BBB and can be detected in the peripheral blood, which provides a minimally invasive method for their detection compared to liquid biopsies obtained from cerebrospinal fluid (CSF). Furthermore, these EVs can be readily distinguished from total murine EVs, since they carry human-specific DNA sequences relevant for GBM biology. In a small cohort of glioma patients, we finally demonstrated that peripheral blood EVs cargo can be successfully used to detect the presence of IDH1G395A, an essential biomarker in the current management of human glioma

Growth of human embryonic stem cells using derivates of human fibroblasts.

The majority of human embryonic stem cell (hESC) lines have been derived and grown using mouse or human feeder cells, or using Matrigel, an animal derivative rich in extracellular matrix (ECM) proteins. However, reliance on feeder layers and animal products limits the manipulation and clinical application of hESC. Alternatively, human fibroblasts produce an ECM which could be employed to coated plates and be easily sterilized. We have shown that hESC grown on this matrix and in the presence of medium conditioned by fibroblast cells maintain markers of pluripotency, including expression of cell surface proteins (SSEA3, SSEA4, TRA-1-60, TRA-1-81), alkaline phosphatase activity, and specific intracellular markers (NANOG, OCT, REX1). Moreover, hESC cultured on this novel human-derived ECM display a normal karyotype. This growth system reduces exposure of hESC to feeder layers and animal ingredients, thereby limiting the risk of pathogenic contamination and additionally facilitating manipulation of hESCs.

Development of a Human Extracellular Matrix for Applications Related with Stem Cells and Tissue Engineering

The recent progress in stem cell biology has created new approaches for their study as well as their application to the treatment of human diseases [1–3]. The success of stem-cell based technologies in the clinical setting [4, 5] has emphasized the need to improve the standards of quality for all phases of cell therapy, particularly the development of culture methods that circumvent products of animal origin since these might provoke infections or immune rejection following transplantation in patients. Indeed, since Martin et al. (2005) [6] demonstrated that hESC cultured with animal or serum products retained non-human sialic acid which was immunogenic when transplanted in humans, the establishment of animal-free conditions to support the maintenance and differentiation of human stem cells has been a major goal of the field of regenerative medicine [7]. Thus, chemically-defined culture systems that are devoid of non-human substances will greatly facilitate the use of stem cells in regenerative strategies. The concept of a niche is crucial for the organization of stem cells. A niche is consider as a subset of tissue cells and extracellular substrates (matrix and soluble factors) that can support stem cells and control their self-renewal in vivo [8]. Extracellular matrices help to structure niches spatially and modulate the concentration of adhesive and signalling molecules locally. The ECM is a molecular complex that contains collagens and other glycoproteins, hyaluronic acid, proteoglycans, glycosaminoglycans (GAGs), and elastins. Additionally, the ECM harbours growth factors or cytokines to protect against degradation [9]. ECM components are responsible for adhesion during the majority of cell interactions and are implicated in the maintenance of embryonic induction during development as well as stem cell differentiation in vitro [10]. Thus, local changes in ECM can dramatically modulate the proliferation and migration of stem cells and may participate in the specification of lineages. hESC have provided invaluable tools for gaining insight into the developmental origins of human tissues. However, to realize the full biological and clinical potential of hESC, certain problems related with the routine culture of these cells must be solved. Mouse embryonic fibroblasts (MEFs) and murine derivatives such as Matrigel are widely used in the maintenance and differentiation of hESC. Recently, considerable effort has been dedicated to the elimination of animal-derived reagents from the culture of hESC and in parallel, to the control of cell growth parameters by avoiding human feeder cells. For example, in 2006 Ludwig et al. reported the use of conditioned media and high doses of FGF to maintain the undifferentiated state in hESC cultured on plastic, but some abnormalities were detected at passage 20 under these conditions [11, 12]. The use of human feeders complicates the growth and molecular analysis of both pluripotency and differentiation since experimental data may reflect the combined effects of hESC and feeder cells in the culture. Given these considerations, the use of an ECM of human or synthetic origin would provide many advantages. Indeed, there have been attempts [13, 14] to produce such a tool but the results have not been satisfactory because the products were unable to maintain hESC in the undifferentiated state over time. Matrix proteins have been used as coating for in vitro cultures of human stem cells but they have usually been applied as undefined protein mixtures [11] of animal origin [15] with undefined media [13], serum [16] or a synthetic mixture [17, 18]. However, most of these human biological reagents are expensive to manufacture and thus, are cost-prohibitive for many laboratories. Decellularization procedures have been used traditionally to isolate ECM from cells in culture, tissues or organs [19]. The goal of decellularization protocols is to efficiently remove cellular and nuclear material while minimizing any adverse effect on the composition, biological activity and mechanical and structural integrity of the remaining ECM [20, 21]. Decellularized human scaffolds have facilitated the remodelling of various tissues in both animal models and humans [4, 22]. However, any biochemical procedure employed to remove cells may also alter the native three-dimensional architecture of the ECM and thus, a balance must be achieved between chemical and physical treatments during the decellularization process [4, 5, 23]. Given the potential importance of hESC in translational research and regenerative medicine, the aim of the present study was to develop a simple, efficient protocol for the production of a human ECM that is both safe and economical. Here we report that hypotonic lysis of human foreskin fibroblasts (HFF) generates a human ECM that retains protein components which are essential for attachment and cell-cell interaction. This hffECM was capable of maintaining the pluripotency of hESC and supporting their differentiation when used with the appropriate medium. Therefore, our results reveal hffECM as a novel tool which may facilitate the clinical application of hESC-based technologies.

Heart valve tissue engineering: how far is the bedside from the bench?

Heart disease, including valve pathologies, is the leading cause of death worldwide. Despite the progress made thanks to improving transplantation techniques, a perfect valve substitute has not yet been developed: once a diseased valve is replaced with current technologies, the newly implanted valve still needs to be changed some time in the future. This situation is particularly dramatic in the case of children and young adults, because of the necessity of valve growth during the patients life. Our review focuses on the current status of heart valve (HV) therapy and the challenges that must be solved in the development of new approaches based on tissue engineering. Scientists and physicians have proposed tissue-engineered heart valves (TEHVs) as the most promising solution for HV replacement, especially given that they can help to avoid thrombosis, structural deterioration and xenoinfections. Lastly, TEHVs might also serve as a model for studying human valve development and pathologies.

Distinct prostate cancer-related mRNA cargo in extracellular vesicle subsets from prostate cell lines

BackgroundMultiple types of extracellular vesicles (EVs), including microvesicles (MVs) and exosomes (EXOs), are released by all cells constituting part of the cellular EV secretome. The bioactive cargo of EVs can be shuffled between cells and consists of lipids, metabolites, proteins, and nucleic acids, including multiple RNA species from non-coding RNAs to messenger RNAs (mRNAs). In this study, we hypothesized that the mRNA cargo of EVs could differ based on the EV cellular origin and subpopulation analyzed.MethodsWe isolated MVs and EXOs from PC-3 and LNCaP prostate cancer cells by differential centrifugation and compared them to EVs derived from the benign PNT2 prostate cells. The relative mRNA levels of 84 prostate cancer-related genes were investigated and validated using quantitative reverse transcription PCR arrays.ResultsBased on the mRNA abundance, MVs rather than EXOs were enriched in the analyzed transcripts, providing a snapshot of the tumor transcriptome. LNCaP MVs specifically contained significantly increased mRNA levels of NK3 Homeobox 1 (NKX3-1), transmembrane protease serine 2 (TMPRSS2), and tumor protein 53 (TP53) genes, whereas PC-3 MVs carried increased mRNA levels of several genes including, caveolin-2 (CAV2), glutathione S-transferase pi 1 (GSTP1), pescadillo ribosomal biogenesis factor 1 (PES1), calmodulin regulated spectrin associated protein 1 (CAMSAP1), zinc-finger protein 185 (ZNF185), and others compared to PNT2 MVs. Additionally, ETS variant 1 (ETV1) and fatty acid synthase (FASN) mRNAs identified in LNCaP- and PC-3- derived MVs highly correlated with prostate cancer progression.ConclusionsOur study provides new understandings of the variability of the mRNA cargo of MVs and EXOs from different cell lines despite same cancer origin, which is essential to better understand the the proportion of the cell transcriptome that can be detected within EVs and to evaluate their role in disease diagnosis.