Functional impairment of human resident cardiac stem cells by the cardiotoxic antineoplastic agent trastuzumab

Trastuzumab (TZM), a monoclonal antibody against the ERBB2 protein, increases survival in ERBB2-positive breast cancer patients. Its clinical use, however, is limited by cardiotoxicity. We sought to evaluate whether TZM cardiotoxicity involves inhibition of human adult cardiac-derived stem cells, in addition to previously reported direct adverse effects on cardiomyocytes. To test this idea, we exposed human cardiosphere-derived cells (hCDCs), a natural mixture of cardiac stem cells and supporting cells that has been shown to exert potent regenerative effects, to TZM and tested the effects in vitro and in vivo. We found that ERBB2 mRNA and protein are expressed in hCDCs at levels comparable to those in human myocardium. Although clinically relevant concentrations of TZM had no effect on proliferation, apoptosis, or size of the c-kit-positive hCDC subpopulation, in vitro assays demonstrated diminished potential for cardiogenic differentiation and impaired ability to form microvascular networks in TZM-treated cells. The functional benefit of hCDCs injected into the border zone of acutely infarcted mouse hearts was abrogated by TZM: infarcted animals treated with TZM + hCDCs had a lower ejection fraction, thinner infarct scar, and reduced capillary density in the infarct border zone compared with animals that received hCDCs alone (n = 12 per group). Collectively, these results indicate that TZM inhibits the cardiomyogenic and angiogenic capacities of hCDCs in vitro and abrogates the morphological and functional benefits of hCDC transplantation in vivo. Thus, TZM impairs the function of human resident cardiac stem cells, potentially contributing to TZM cardiotoxicity

Phylogenetic analysis of planarian collagens and their roles in regeneration

Poster Presentation - Theme 3: Development & stem cellsStem cells are regulated by the microenvironment or niche they reside in, which consists of growth factors, niche cells and the extracellular matrix. The ECM acts as both a structural component and as a reservoir for growth factors that are released upon degradation. During regeneration, stem cells in the planarian are activated to migrate and proliferate; however, the role of the ECM in stem cell regulation is still unclear. Analysis of an EST library of planarian transcripts revealed nine fibrillar-related collagen chains (DjCol1-9). Sequence and structural analysis ...postprin

Isolation of Human Photoreceptor Precursors via a Cell Surface Marker Panel from Stem Cell-derived Retinal Organoids and Fetal Retinae

Loss of photoreceptor cells due to retinal degeneration is one of the main causes of blindness in the developed world. Although there is currently no effective treatment, cell replacement therapy using stem-cell-derived photoreceptor cells may be a feasible future treatment option. In order to ensure safety and efficacy of this approach, robust cell isolation and purification protocols must be developed. To this end, we previously developed a biomarker panel for the isolation of mouse photoreceptor precursors from the developing mouse retina and mouse embryonic stem cell cultures. In the current study we applied this approach to the human pluripotent stem cell (hPSC) system, and identified novel biomarker combinations that can be leveraged for the isolation of human photoreceptors. Human retinal samples and hPSC-derived retinal organoid cultures were screened against 242 human monoclonal antibodies using a high through-put flow cytometry approach. We identified 46 biomarkers with significant expression levels in the human retina and hPSC differentiation cultures. Human retinal cell samples, either from fetal tissue or derived from embryonic and induced pluripotent stem cell cultures, were FAC-sorted using selected candidate biomarkers that showed expression in discrete cell populations. Enrichment for photoreceptors and exclusion of mitotically active cells was demonstrated by immunocytochemical analysis with photoreceptor-specific antibodies and Ki-67. We established a biomarker combination, which enables the robust purification of viable human photoreceptors from both human retinae and hPSC-derived organoid cultures. This article is protected by copyright. All rights reserved

Differential regulation of human bone marrow mesenchymal stromal cell chondrogenesis by hypoxia inducible factor-1α hydroxylase inhibitors

The transcriptional profile induced by hypoxia plays important roles in the chondrogenic differentiation of marrow stromal/stem cells (MSC) and is mediated by the Hypoxia Inducible Factor complex. However, various compounds can also stabilise HIF's oxygen-responsive element, HIF-1α, at normoxia and mimic many hypoxia-induced cellular responses. Such compounds may prove efficacious in cartilage tissue engineering, where microenvironmental cues may mediate functional tissue formation. Here, we investigated three HIF stabilising compounds, which each have distinct mechanisms of action, to understand how they differentially influenced the chondrogenesis of human bone marrow-derived MSC (hBM-MSC) in vitro. hBM-MSCs were chondrogenically-induced in TGF-β3 -containing media in the presence of HIF-stabilising compounds. HIF-1α stabilisation was assessed by HIF-1α immunofluorescence staining, expression of HIF target and articular chondrocyte specific genes by qPCR, and cartilage-like extracellular matrix (ECM) production by immunofluorescence and histochemical staining. We demonstrate that all three compounds induced similar levels of HIF-1α nuclear localisation. However, whilst the 2-oxoglutarate analogue Dimethyloxalylglycine (DMOG) promoted upregulation of a selection of HIF target genes, Desferrioxamine (DFX) and Cobalt Chloride (CoCl2 ), compounds that chelate or compete with Fe2+ , respectively, did not. Moreover, DMOG induced a more chondrogenic transcriptional profile, which was abolished by Acriflavine, an inhibitor of HIF-1α-HIF-β binding, whilst the chondrogenic effects of DFX and CoCl2 were more limited. Together, these data suggest that HIF-1α function during hBM-MSC chondrogenesis may be regulated by mechanisms with a greater dependence on 2-oxoglutarate than Fe2+ availability. These results may have important implications for understanding cartilage disease and developing targeted therapies for cartilage repair. This article is protected by copyright. All rights reserved

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

A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence.

We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies

The Stem Cell Microenvironment and Its Role in Regenerative Medicine and Cancer Pathogenesis

How stem cells behave is very much a factor of their local microenvironment, also known as the stem cell niche. Physical, chemical, or electrical signals from the neighboring cells or biochemical signals from distant cells are crucial in the cell fate decision process. A major challenge of tissue engineering is to mimic the natural cell environment by designing very sophisticated scaffolds able not only to mechanically support cells, but also to release signals biologically relevant for governing stem cell fate. In addition, increasing evidence suggests that abnormal interaction of stem cells with their niche is responsible for altered cell function leading to malignant transformation. This book discusses some of the recent advances in stem cell research that may help understanding the properties of the niche that govern stem cell fate. Technical topics discussed include:Stem cell biologyCancer stem cellsStem cell interactions with biomaterialsEngineering the stem cell microenvironmentStem cells in tissue regeneration and repairThe Disputationes Workshop series is an international initiative aimed at disseminating stem cell related cutting edge knowledge among scientists, healthcare workers, students and policy makers. This book emerges as a result of the scientific contributions presented and discussed during the fifth Disputationes Workshop held in Aalborg (Denmark) in April 2014. The stem cell microenvironment and its role in regenerative medicine and cancer pathogenesis is ideal for academic staff and master/research students in biomedical and health science

Identification of Multipotent Progenitors that Emerge Prior to Hematopoietic Stem Cells in Embryonic Development

Summary Hematopoiesis in the embryo proceeds in a series of waves, with primitive erythroid-biased waves succeeded by definitive waves, within which the properties of hematopoietic stem cells (multilineage potential, self-renewal, and engraftability) gradually arise. Whereas self-renewal and engraftability have previously been examined in the embryo, multipotency has not been thoroughly addressed, especially at the single-cell level or within well-defined populations. To identify when and where clonal multilineage potential arises during embryogenesis, we developed a single-cell multipotency assay. We find that, during the initiation of definitive hematopoiesis in the embryo, a defined population of multipotent, engraftable progenitors emerges that is much more abundant within the yolk sac (YS) than the aorta-gonad-mesonephros (AGM) or fetal liver. These experiments indicate that multipotent cells appear in concert within both the YS and AGM and strongly implicate YS-derived progenitors as contributors to definitive hematopoiesis

Stem cells and cancer stem cells

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