Joakim Sandstedt

C-kit+ CD45- cells found in the adult human heart represent a population of endothelial progenitor cells.

Although numerous reports support the existence of stem cells in the adult heart, few studies have been conducted using human cardiac tissue. Therefore, cells from human cardiac atrial biopsies were analyzed regarding progenitor properties. Expression of stem cell markers was analyzed using fluorescence-activated cell sorting. This identified a small population of C-kit+ cells, which could be further subdivided based on expression of CD45. The C-kit+ CD45+ population was determined to be of mast cell identity, while the C-kit+ CD45− population expressed mRNA of the endothelial lineage. Since the number of cells obtainable from biopsies was limited, a comparison between directly isolated and monolayer and explant cultured cells, respectively, was carried out. While both cultures retained a small population of mast cells, only monolayer culture produced a stable and relatively high percentage of C-kit+ CD45− cells. This population was found to co-express endothelial progenitor cell markers such as CD31, CD34, CXCR4, and FLK-1. The mRNA expression profile was similar to the one from directly isolated cells. When sorted cells were cultured in endothelial differentiation medium, the C-kit+ CD45− population retained its expression of endothelial markers to a large extent, but downregulated progenitor markers, indicating further differentiation into endothelial cells. We have confirmed that the human cardiac atrium contains a small C-kit+ CD45− population expressing markers commonly found on endothelial progenitor cells. The existence of an endothelial progenitor population within the heart might have future implications for developing methods of inducing neovascularization after myocardial infarction.

Neither Notch1 Expression nor Cellular Size Correlate with Mesenchymal Stem Cell Properties of Adult Articular Chondrocytes

Background: Tissue repair is thought to be regulated by progenitor cells, which in other tissues are characterized by their Notch1 expression or small cellular size. Here we studied if these traits affect the chondrogenic potential and are markers for multipotent progenitor cell populations in adult articular cartilage. Methods: Directly isolated articular chondrocytes were sorted with regard to their Notch1 expression or cellular size. Their colony forming efficiency (CFE) and their potential to differentiate towards adipogenic, osteogenic and chondrogenic lineages were investigated. The different sorted populations were also expanded in monolayer and analyzed in the same manner as the directly isolated cells. Results: No differences in CFE or adipogenic, osteogenic and chondrogenic potentials were detected among the sorted populations. Expanded cells displayed a higher osteochondral potential than directly isolated cells. Conclusion: Cellular size or Notch1 expression is not per se a specific marker for mesenchymal progenitor cells in adult articular cartilage. Monolayer-expanded adult chondrocytes contain a larger mesenchymal progenitor cell-like population than directly isolated cells, highly likely as a result of dedifferentiation. If there are resident Notch1-positive cells or cells of a specific size in adult articular cartilage with functional features of progenitor cells, the population consists of only a very small number of cells.

Human C-kit+CD45- cardiac stem cells are heterogeneous and display both cardiac and endothelial commitment by single-cell qPCR analysis.

C-kit expressing cardiac stem cells have been described as multipotent. We have previously identified human cardiac C-kit+CD45- cells, but only found evidence of endothelial commitment. A small cardiac committed subpopulation within the C-kit+CD45- population might however be present. To investigate this at single-cell level, right and left atrial biopsies were dissociated and analyzed by FACS. Only right atrial biopsies contained a clearly distinguishable C-kit+CD45- population, which was single-cell sorted for qPCR. A minor portion of the sorted cells (1.1%) expressed early cardiac gene NKX2.5 while most of the cells (81%) expressed late endothelial gene VWF. VWF- cells were analyzed for a wider panel of genes. One group of these cells expressed endothelial genes (FLK-1, CD31) while another group expressed late cardiac genes (TNNT2, ACTC1). In conclusion, human C-kit+CD45- cells were predominantly localized to the right atrium. While most of these cells expressed endothelial genes, a minor portion expressed cardiac genes.

Combination of positive charges and honeycomb pores to promote MC3T3-E1 cell behaviour

A facile chemistry route to prepare symmetric poly(L-lactide) (PLLA)-based dendritic L-lysine copolymer (PLLA-d), with a PLLA block as the core and lysine dendrons in the two ends to provide certain density of positive charges, through a divergent method is reported. The polymers were characterized by H-1 NMR, GPC and MALDI-TOF to confirm the well-defined chemical architecture. The study on crystallization behaviour demonstrated that the introduction of the lysine dendron favoured the formation of banded spherulites when compared with the PLLA polymer. The differential scanning calorimetry (DSC) results showed that the lysine dendron disrupted PLLA crystalline region and lowered the melting point and crystallinity of PLLA. The PLLA-d was fabricated into honeycomb films (H-PLLA-d) through the breath-figure method for water contact angle test and in vitro study, with flat PLLA, honeycomb PLLA, and flat PLLA-d films (PLLA, H-PLLA, and F-PLLA-d, respectively) as the controls. The water contact angle test indicated that the hydrophilicity of the PLLA-d film was strongly improved after the incorporation of the lysine dendron into PLLA. The incorporation of the lysine dendron increased the surface zeta potential and decreased the mechanical properties of PLLA. Mouse osteoblastic cell (MC3T3-E1) functions including cell attachment, adhesion, proliferation, and differentiation were investigated on PLLA, H-PLLA, F-PLLA-d and H-PLLA-d films. The results indicated that MC3T3-E1 cell functions were significantly enhanced on F-PLLA-d or H-PLLA films and especially H-PLLA-d ones. This study not only demonstrates a facile approach to fabricate a novel copolymer film (H-PLLA-d), which combines positive charges with honeycomb pores, but also provides a potential biomaterial for bone repair by improving osteoblastic cell functions.

SSEA-4+ CD34- Cells in the Adult Human Heart Show the Molecular Characteristics of a Novel Cardiomyocyte Progenitor Population

Stage-specific embryonic antigen (SSEA) expression is used to describe the differentiation state of an embryonic stem cell (ESC). In human ESCs, SSEA-3 and SSEA-4 are highly expressed in undifferentiated cells and downregulated upon differentiation. SSEA-4 has also been described as a marker for adult stem cells in various tissues, including human neonatal cardiac tissue. However, there is currently little data on the expression of SSEAs in human adult cardiac tissue. We obtained right and left atrial biopsies from patients undergoing cardiac surgery. These were dissociated, stained for SSEAs and other cardiac stem cell markers and analyzed by flow cytometry. Directly isolated cells expressed variable levels of SSEA-1, SSEA-3 and SSEA-4. The SSEA-1+ population was established as contaminating hematopoietic cells. The SSEA-4+ population, on the other hand, could be subdivided based on the endothelial progenitor marker CD34. The SSEA-4+ CD34- population in the right atrium had a high gene expression of both early (TBX5, NKX2.5) and late (TNNT2) cardiomyocyte markers. The SSEA-4+ CD34+ population, on the other hand, overlapped with previously described C-kit+ CD45- cardiac stem cells. Primary monolayer-cultured cells retained expression of SSEAs while the cardiomyogenic specification in the SSEA-4+ CD34- population was lost. In tissue sections, SSEA-4+ cells could be identified both within and outside the myocardium. Within the myocardium, some SSEA-4+ cells coexpressed cardiomyogenic markers. In conclusion, the results show that the adult human heart expresses SSEAs and that there is a subpopulation of SSEA-4+ CD34- cells that show features of a cardiomyocyte progenitor population.

The Arachidonate 15-Lipoxygenase Enzyme Product 15-HETE Is Present in Heart Tissue from Patients with Ischemic Heart Disease and Enhances Clot Formation.

Ischemic heart disease is a major cause of death and morbidity and the search for novel therapeutic targets is still required. We have previously shown that the enzyme arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), is highly expressed in ischemic heart tissue, but its role in the pathogenesis of ischemic heart disease is unclear. Here we showed that expression of ALOX15, but not ALOX12 or ALOX15B, was increased in ischemic versus non-ischemic human heart biopsy samples. A similar ALOX expression pattern was found in hypoxic human cardiomyocytes and cardiac endothelial cells. We also showed that levels of 15-HETE were significantly higher in ischemic versus non-ischemic human heart biopsy samples and showed a tendency to increase in serum from the patients with ischemic heart disease. Moreover, hypoxia increased the production of 15-HETE levels from human cardiomyocytes and cardiac endothelial cells. The hypoxia-induced increase in 15-HETE levels from human cardiomyocytes was inhibited by the ALOX15 inhibitor baicalein. Finally, by using intrinsic rotational thromboelastometry, we showed that human whole blood clotted faster in the presence of 15-HETE. In summary, we propose that increased ALOX15 expression in heart tissue under ischemic conditions may lead to increased production of 15-HETE, potentially contributing to thrombosis.

Intracellular flow cytometry may be combined with good quality and high sensitivity RT‐qPCR analysis

Flow cytometry (FCM) has become a well‐established method for analysis of both intracellular and cell‐surface proteins, while quantitative RT‐PCR (RT‐qPCR) is used to determine gene expression with high sensitivity and specificity. Combining these two methods would be of great value. The effects of intracellular staining on RNA integrity and RT‐qPCR sensitivity and quality have not, however, been fully examined. We, therefore, intended to assess these effects further. Cells from the human lung cancer cell line A549 were fixed, permeabilized and sorted by FCM. Sorted cells were analyzed using RT‐qPCR. RNA integrity was determined by RNA quality indicator analysis. A549 cells were then mixed with cells of the mouse cardiomyocyte cell line HL‐1. A549 cells were identified by the cell surface marker ABCG2, while HL‐1 cells were identified by intracellular cTnT. Cells were sorted and analyzed by RT‐qPCR. Finally, cell cultures from human atrial biopsies were used to evaluate the effects of fixation and permeabilization on RT‐qPCR analysis of nonimmortalized cells stored prior to analysis by FCM. A large amount of RNA could be extracted even when cells had been fixed and permeabilized. Permeabilization resulted in increased RNA degradation and a moderate decrease in RT‐qPCR sensitivity. Gene expression levels were also affected to a moderate extent. Sorted populations from the mixed A549 and HL‐1 cell samples showed gene expression patterns that corresponded to FCM data. When samples were stored before FCM sorting, the RT‐qPCR analysis could still be performed with high sensitivity and quality. In summary, our results show that intracellular FCM may be performed with only minor impairment of the RT‐qPCR sensitivity and quality when analyzing sorted cells; however, these effects should be considered when comparing RT‐qPCR data of not fixed samples with those of fixed and permeabilized samples.

Triphasic and quadriphasic waveforms are superior to biphasic waveforms for synchronized beating of cardiomyocytes

BACKGROUND AND PURPOSE Within pacemaker research few attempts have been made to find an optimal waveform phase sequence that synchronizes beating of cardiomyocytes at an electrode. Multielectrode arrays (MEAs) offer electrophysiological screening of cardiomyocytes serving as a system for preliminary screening of pacing waveform design. MATERIALS AND METHODS The HL-1 cell line was cultured in MEAs until confluence and stimulated with biphasic, triphasic, and quadriphasic waveforms. The amplitudes required for synchronized beating of the cells were determined. RESULTS Triphasic and quadriphasic waveforms were more efficient in eliciting synchronized beating of the HL-1 cells compared with the biphasic waveform because it allows significant reductions in synchronizing voltage amplitudes and reductions in supplied stimulus. CONCLUSION The MEA system allows for a straightforward manner to investigate effects of waveform design on synchronized beating in cardiomyocytes in vitro. Increased number of phase changes in a pacing waveform seems to be the major reason for the reduction in synchronizing amplitudes.

Hypoxic cardiac fibroblasts from failing human hearts decrease cardiomyocyte beating frequency in an ALOX15 dependent manner

A common denominator for patients with heart failure is the correlation between elevated serum levels of proinflammatory cytokines and adverse clinical outcomes. Furthermore, lipoxygenase-induced inflammation is reportedly involved in the pathology of heart failure. Cardiac fibroblasts, which are abundant in cardiac tissue, are known to be activated by inflammation. We previously showed high expression of the lipoxygenase arachidonate 15 lipoxygenase (ALOX15), which catalyzes the conversion of arachidonic acid to 15-hydroxy eicosatetraenoic acid (15-HETE), in ischemic cardiac tissue. The exact roles of ALOX15 and 15-HETE in the pathogenesis of heart failure are however unknown. Biopsies were collected from all chambers of explanted failing human hearts from heart transplantation patients, as well as from the left ventricles from organ donors not suffering from chronic heart failure. Biopsies from the left ventricles underwent quantitative immunohistochemical analysis for ALOX15/B. Gene expression of ALOX enzymes, as well as 15-HETE levels, were examined in cardiac fibroblasts which had been cultured in either hypoxic or normoxic conditions after isolation from failing hearts. After the addition of fibroblast supernatants to human induced pluripotent stem cell-derived cardiomyocytes, intracellular calcium concentrations were measured to examine the effect of paracrine signaling on cardiomyocyte beating frequency. While ALOX15 and ALOX15B were expressed throughout failing hearts as well as in hearts from organ donors, ALOX15 was expressed at significantly higher levels in donor hearts. Hypoxia resulted in a significant increase in gene and protein expression of ALOX15 and ALOX15B in fibroblasts isolated from the different chambers of failing hearts. Finally, preconditioned medium from hypoxic fibroblasts decreased the beating frequency of human cardiomyocytes derived from induced pluripotent stem cells in an ALOX15-dependent manner. In summary, our results demonstrate that ALOX15/B signaling by hypoxic cardiac fibroblasts may play an important role in ischemic cardiomyopathy, by decreasing cardiomyocyte beating frequency.

Human cardiac fibroblasts isolated from patients with severe heart failure are immune-competent cells mediating an inflammatory response

&NA; This study was aimed to elucidate the immunoregulatory properties of human cardiac fibroblasts cultured under pro‐inflammatory and hypoxic conditions. Human heart tissue for isolating cardiac cells is generally hard to obtain, particularly from all four chambers of the same heart. Since different parts of the heart have different functions and therefore may have different immunoregulatory properties, ability to analyse cells from all chambers allows for a unique and comprehensive investigation. Cells were isolated from all four chambers of the heart from patients undergoing cardiac transplantation surgery due to severe chronic heart failure (CHF) (n = 6). Cells isolated from one donor heart, were used for comparison with the experimental group. Primary cultured human cardiac fibroblasts were treated with Lipopolysaccharide (LPS) to induce an inflammatory response. Cells were also subjected to hypoxia. To determine immunoregulatory properties of the cells, cytokine and chemokine profiles were determined using multiplex ELISA. Results: On average, the fibroblasts population constituted approximately 90% of the expanded non‐myocytes. Levels of cytokines and chemokines were markedly increased in human cardiac fibroblasts cultured under inflammatory conditions, with a similar response in fibroblasts from all compartments of the heart. Unexpectedly, hypoxia did not further augment cytokine and chemokine secretion. In conclusion, human cardiac fibroblasts are a robust source of pro‐inflammatory mediators in the failing heart, independent of hypoxia, and might play a critical role in inflammation associated with the pathogenesis of CHF.