Rafael Kramann

Perivascular Gli1+ Progenitors Are Key Contributors to Injury-Induced Organ Fibrosis

Mesenchymal stem cells (MSCs) reside in the perivascular niche of many organs, including kidney, lung, liver, and heart, although their roles in these tissues are poorly understood. Here, we demonstrate that Gli1 marks perivascular MSC-like cells that substantially contribute to organ fibrosis. In vitro, Gli1(+) cells express typical MSC markers, exhibit trilineage differentiation capacity, and possess colony-forming activity, despite constituting a small fraction of the platelet-derived growth factor-β (PDGFRβ)(+) cell population. Genetic lineage tracing analysis demonstrates that tissue-resident, but not circulating, Gli1(+) cells proliferate after kidney, lung, liver, or heart injury to generate myofibroblasts. Genetic ablation of these cells substantially ameliorates kidney and heart fibrosis and preserves ejection fraction in a model of induced heart failure. These findings implicate perivascular Gli1(+) MSC-like cells as a major cellular origin of organ fibrosis and demonstrate that these cells may be a relevant therapeutic target to prevent solid organ dysfunction after injury.

Analysis of myocardial deformation based on pixel tracking in two dimensional echocardiographic images enables quantitative assessment of regional left ventricular function

Objective: To evaluate whether myocardial strain and strain rate calculated from two dimensional echocardiography by automatic frame-by-frame tracking of natural acoustic markers enables objective description of regional left ventricular (LV) function. Methods: In 64 patients parasternal two dimensional echocardiographic views at the apical, mid-ventricular and basal levels were obtained. An automatic frame-by-frame tracking system of natural acoustic echocardiographic markers was used to calculate radial strain, circumferential strain, radial strain rate and circumferential strain rate for each LV segment in a 16 segment model. Cardiac magnetic resonance imaging (cMRI) was performed to define segmental LV function as normokinetic, hypokinetic or akinetic. Results: Image quality was sufficient for adequate strain and strain-rate analysis from two dimensional echocardiographic images obtained from parasternal views in 88% of segments. Obtained radial strain data were highly reproducible and analysis was affected by only small intraobserver (mean 4.4 (SD 1.6)%) and interobserver variabilities (7.3 (2.5)%). Each of the analysed strain and strain-rate parameters was significantly different between segments defined as normokinetic, hypokinetic or akinetic by cMRI (radial strain 36.8 (10.5)%, 24.1 (7.5)% and 13.4 (4.8)%, respectively, p < 0.001). Peak systolic radial strain enabled detection of hypokinesis or akinesis with a sensitivity of 83.5% and a specificity of 83.5% (cut off value 29.1%, receiver operating characteristic (ROC) curve area 0.905, 95% CI 0.883 to 0.923). Peak systolic radial strain analysis also enabled detection of akinesis versus hypokinesis with a sensitivity of 82.7% and a specificity of 94.5% (cut off value 21.0%, ROC curve area 0.946). Peak systolic radial strain-rate analysis was less accurate than peak systolic radial strain analysis to detect cMRI-defined segmental function abnormalities. The accuracy of peak systolic circumferential strain and strain rate was similar to that of corresponding radial parameters. Conclusions: Frame-by-frame tracking of acoustic markers in two dimensional echocardiographic images enables accurate analysis of regional systolic LV function.

Differentiated kidney epithelial cells repair injured proximal tubule

Significance When epithelial cells in the proximal portion of the nephron are damaged they rapidly proliferate to repair the damage to the kidney. Whether a stem cell is responsible for this proliferative response or not is controversial. Although a scattered population of cells can be found in the human proximal tubule that seem to have stem-cell characteristics, they could also represent isolated damaged cells that have dedifferentiated and lost their epithelial characteristics. We resolve these conflicting models using genetic lineage analysis to demonstrate that fully differentiated proximal tubule cells not only proliferate after injury, but they also upregulate apparent stem-cell markers. This study shows that epithelial dedifferentiation is responsible for repair of mouse proximal tubule, rather than an adult stem-cell population. Whether kidney proximal tubule harbors a scattered population of epithelial stem cells is a major unsolved question. Lineage-tracing studies, histologic characterization, and ex vivo functional analysis results conflict. To address this controversy, we analyzed the lineage and clonal behavior of fully differentiated proximal tubule epithelial cells after injury. A CreERT2 cassette was knocked into the sodium-dependent inorganic phosphate transporter SLC34a1 locus, which is expressed only in differentiated proximal tubule. Tamoxifen-dependent recombination was absolutely specific to proximal tubule. Clonal analysis after injury and repair showed that the bulk of labeled cells proliferate after injury with increased clone size after severe compared with mild injury. Injury to labeled proximal tubule epithelia induced expression of CD24, CD133, vimentin, and kidney-injury molecule-1, markers of putative epithelial stem cells in the human kidney. Similar results were observed in cultured proximal tubules, in which labeled clones proliferated and expressed dedifferentiation and injury markers. When mice with completely labeled kidneys were subject to injury and repair there was no dilution of fate marker despite substantial proliferation, indicating that unlabeled progenitors do not contribute to kidney repair. During nephrogenesis and early kidney growth, single proximal tubule clones expanded, suggesting that differentiated cells also contribute to tubule elongation. These findings provide no evidence for an intratubular stem-cell population, but rather indicate that terminally differentiated epithelia reexpress apparent stem-cell markers during injury-induced dedifferentiation and repair.

The osteogenic differentiation of adult bone marrow and perinatal umbilical mesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds.

Adult human mesenchymal stem cells from bone marrow (BM-MSC) represent a promising source for skeletal regeneration. Perinatal MSC from Whartons Jelly of the umbilical cord (UC-MSC) are expected to possess enhanced differentiation capacities due to partial expression of pluripotency markers. For bone tissue engineering, it is important to analyse in vitro behaviour of stem cell/biomaterial hybrids concerning in vivo integration into injured tissue via migration, matrix remodelling and differentiation. This study compares the cell-mediated remodelling of three-dimensional collagen I/III gels during osteogenic differentiation of both cell types. When activated through collagen contact and subjected to osteogenic differentiation, UC-MSC differ from BM-MSC in expression and synthesis of extracellular matrix (ECM) proteins as shown by histology, immunohistochemistry, Western Blot analysis and realtime-RT-PCR. The biosynthetic activity was accompanied in both cell types by the ultrastructural appearance of hydroxyapatite/calcium crystals and osteogenic gene induction. Following secretion of matrix metalloproteinases (MMP), both MSC types migrated into and colonised the collagenous matrix causing matrix strengthening and contraction. These results indicate that UC-MSC and BM-MSC display all features needed for effective bone fracture healing. The expression of ECM differs in both cell types considerably, suggesting different mechanisms for bone formation and significant impact for bone tissue engineering.

Impact of left ventricular lead position on the efficacy of cardiac resynchronisation therapy: a two-dimensional strain echocardiography study.

Background: Definition of the optimal left ventricular (LV) lead position in cardiac resynchronisation therapy (CRT) is desirable. Objective: To define the optimal LV lead position in CRT and assess the effectiveness of CRT depending on the LV lead position using new myocardial deformation imaging. Methods: Myocardial deformation imaging based on tracking of acoustic tissue pixels in two-dimensional echocardiographic images (EchoPAC, GE ultrasound) was performed in 47 patients with heart failure at baseline and during CRT. In a 36-segment LV model the segment with the latest peak systolic circumferential strain before CRT was determined. The segment with maximal temporal difference in peak systolic circumferential strain on CRT compared with before CRT was assumed to be the LV lead position. The optimal LV lead position was defined as concurrence or immediate neighbouring of the segment with the latest contraction before CRT and those with assumed LV lead location. Results: 25 patients had optimal and 22 non-optimal LV lead positions. Before CRT, the LV ejection fraction (EF) and peak oxygen consumption (Vo2max) were similar in patients with optimal and non-optimal LV lead positions (mean (SD) EF = 31.4 (6.1)% vs 30.3 (6.5)% and Vo2max = 14.2 (1.8) vs 14.0 (2.1) ml/min/kg, respectively). At 3 months on CRT, EF increased by 9 (2)% vs 5 (3)% and Vo2max by 2.0 (0.8) vs 1.1 (0.5) ml/min/kg in the optimal vs non-optimal LV lead position groups, respectively (both p<0.001). Conclusions: Concordance of the LV lead site and location of the latest systolic contraction before CRT results in greater improvement in EF and cardiopulmonary workload than the non-optimal LV lead position.

Understanding the origin, activation and regulation of matrix‐producing myofibroblasts for treatment of fibrotic disease

Fibrosis and scar formation results from chronic progressive injury in virtually every tissue and affects a growing number of people around the world. Myofibroblasts drive fibrosis, and recent work has demonstrated that mesenchymal cells, including pericytes and perivascular fibroblasts, are their main progenitors. Understanding the cellular mechanisms of pericyte/fibroblast‐to‐myofibroblast transition, myofibroblast proliferation and the key signalling pathways that regulate these processes is essential to develop novel targeted therapeutics for the growing patient population suffering from solid organ fibrosis. In this review, we summarize the current knowledge about different progenitor cells of myofibroblasts, discuss major pathways that regulate their transdifferentiation and discuss the current status of novel targeted anti‐fibrotic therapeutics in development. Copyright

3D co-culture of hematopoietic stem and progenitor cells and mesenchymal stem cells in collagen scaffolds as a model of the hematopoietic niche.

Here, we propose a collagen-based three-dimensional (3D) environment for hematopoietic stem and progenitor cells (HPC) with mesenchymal stem cells (MSC) derived either from bone marrow (BM) or umbilical cord (UC), to recapitulate the main components of the BM niche. Mechanisms described for HPC homeostasis were systematically analyzed in comparison to the conventional liquid HPC culture. The 3D-cultivation allows dissecting two sub-populations of HPC: (I) HPC in suspension above the collagen gel and (II) migratory HPC in the collagen fibres of the collagen gel. The different sites represent distinct microenvironments with significant impact on HPC fate. HPC in niche I (suspension) are proliferative and a dynamic culture containing HPC (CD34(+)/CD38(-)), maturing myeloid cells (CD38(+), CD13(+), CAE(+)) and natural killer (NK) cells (CD56(+)). In contrast, HPC in niche II showed clonal growth with significant high levels of the primitive CD34(+)/CD38(-) phenotype with starting myeloid (CD13(+), CAE(+)) differentiation, resembling the endosteal part of the BM niche. In contrast, UC-MSC are not adequate for HSC expansion as they significantly enhance HPC proliferation and lineage commitment. In conclusion, the 3D-culture system using collagen and BM-MSC enables HPC expansion and provides a potential platform to dissect regulatory mechanisms in hematopoiesis.

Role of casein kinase 1A1 in the biology and targeted therapy of del(5q) MDS.

The casein kinase 1A1 gene (CSNK1A1) is a putative tumor suppressor gene located in the common deleted region for del(5q) myelodysplastic syndrome (MDS). We generated a murine model with conditional inactivation of Csnk1a1 and found that Csnk1a1 haploinsufficiency induces hematopoietic stem cell expansion and a competitive repopulation advantage, whereas homozygous deletion induces hematopoietic stem cell failure. Based on this finding, we found that heterozygous inactivation of Csnk1a1 sensitizes cells to a CSNK1 inhibitor relative to cells with two intact alleles. In addition, we identified recurrent somatic mutations in CSNK1A1 on the nondeleted allele of patients with del(5q) MDS. These studies demonstrate that CSNK1A1 plays a central role in the biology of del(5q) MDS and is a promising therapeutic target.

Myocardial Deformation Imaging Based on Ultrasonic Pixel Tracking to Identify Reversible Myocardial Dysfunction

OBJECTIVES This study evaluated the predictive value of myocardial deformation imaging for improvement in cardiac function after revascularization therapy in comparison with contrast-enhanced cardiac magnetic resonance imaging (ceMRI). BACKGROUND Myocardial deformation imaging allows analysis of myocardial viability in ischemic left ventricular dysfunction. METHODS In 53 patients with ischemic left ventricular dysfunction, myocardial viability was assessed using pixel-tracking-derived myocardial deformation imaging and ceMRI to predict recovery of function at 9 +/- 2 months follow-up. For each left ventricular segment in a 16-segment model, peak systolic radial strain was determined from parasternal 2-dimensional echocardiographic views using an automatic frame-by-frame tracking system of natural acoustic echocardiographic markers (EchoPAC, GE Ultrasound, Horton, Norway), and the relative extent of hyperenhancement using ceMRI. RESULTS Of 463 segments with abnormal baseline function, 227 showed regional recovery. Compared with segments showing functional improvement, those that failed to recover had lower peak radial strain (15.2 +/- 7.5% vs. 22.6 +/- 6.3%; p < 0.001) and a greater extent of hyperenhancement (56 +/- 29% vs. 14 +/- 17%; p < 0.001). Using a cutoff of 17.2% for peak systolic radial strain, functional recovery could be predicted with high accuracy (sensitivity 70.2%, specificity 85.1%, area under the curve 0.859, 95% confidence interval 0.825 to 0.893). The predictive value was similar to that of hyperenhancement by ceMRI (sensitivity 71.6%, specificity 92.1%, area under the curve 0.874, 95% confidence interval 0.840 to 0.901, at a cutoff of 43% hyperenhancement). CONCLUSIONS Myocardial deformation imaging based on frame-to-frame tracking of acoustic markers in 2-dimensional echocardiographic images is a powerful novel modality to identify reversible myocardial dysfunction.

Long-term survival and characterisation of human umbilical cord-derived mesenchymal stem cells on dermal equivalents.

During early embryogenesis, mesenchymal cells arise from the primitive epithelium and can revert to an epithelial phenotype by passing through mesenchymal-to-epithelial transition (MET). Mesenchymal stem cells (MSC) of the Whartons Jelly of the umbilical cord (UC-MSC) express pluripotency markers underlining their primitive developmental state. As mesenchymal stem cells from bone marrow (BM-MSC) possess a strong propensity to ameliorate mesenchymal tissue damage, UC-MSC might also be able to differentiate into cells apart from the mesoderm, allowing replacement of ectodermal and mesodermal tissues. In this study, we analysed the possible epidermal differentiation of UC-MSC on dermal equivalents (DEs) consisting of collagen I/III with dermal fibroblasts and subjected to the culture conditions for tissue engineering of skin with keratinocytes. The culture conditions were further modified by pre-treating the cells with 5-azacytidine or by supplementing the medium with all trans retinoic acid. Interestingly, a subpopulation of UC-MSC (29%) co-expressed pan-cytokeratin (epithelial marker; pan-CK) and vimentin (mesenchymal marker) after isolation. Under the three-dimensional conditions of skin, the number of pan-CK(+)-cells increased to >30% after 21 days of cultivation, while under osteogenic culture conditions the cells were pan-CK-negative, thus showing the influence of the artificial niche. Nevertheless, the pan-CK-expression was neither accompanied by typical epithelial morphology nor expression of other epidermal markers. The pan-CK-detection can be explained by the expression of cytokeratins in myofibroblasts. UC-MSC expressed alpha-smooth muscle actin after isolation and displayed all features of functional myofibroblasts like morphology, cell-mediated contraction of a collagen gel and production of components of the extracellular matrix (ECM). The treatment with all trans retinoic acid or 5-azacytidine could neither induce an epidermal differentiation nor enhance the myofibroblastic differentiation. Concluding, UC-MSC might be an interesting cell source to support the regeneration of wounds by their differentiation into myofibroblasts and their extensive synthesis of ECM components.