Sca-1+ endothelial cells (SPECs) reside in the portal area of the liver and contribute to rapid recovery from acute liver disease

The liver has a high potential to regenerate however, the relation between oval cells and endothelial cells in the portal area during liver regeneration has not been adequately described. We have focused on sca-1+ endothelial cells (SPEC: sca-1+CD31+CD45− cells) and analyzed their localization, growth potential, and the role of these cells in damaged liver. SPECs are localized in the portal area and comprise approximately 20-30% of CD31+CD45− cells. These cells have higher growth potential than sca-1− endothelial cells and grow aggressively when the liver is severely damaged on the lateral side of the oval cells. In an in vivo study we show that when the liver is severely damaged in the presence of a VEGF (vascular endothelial growth factor)-inhibitor, the frequency of SPECs decreased and the recovery of liver volume was also delayed. These results strongly suggest that SPECs play important roles in the recovery of severely damaged liver.     

Phenotypic characterization of mammosphere-forming cells from the human MA-11 breast carcinoma cell line

The phenotypic diversity of breast carcinoma may be explained by the existence of a sub-population of breast cancer cells, endowed with stem cell-like properties and gene expression profiles, able to differentiate along different pathways. A stem cell-like population of CD44⁺CD24^−/low breast cancer cells was originally identified using cells from metastatic pleural effusions of breast carcinoma patients. We have previously reported that upon in vitro culture as mammospheres under stem cell-like conditions, human MA-11 breast carcinoma cells acquired increased tumorigenicity and lost CD24 expression compared with the parental cell line. We now report that upon passage of MA-11 mammospheres into serum-supplemented cultures, CD24 expression was restored; the rapid increase in CD24 expression was consistent with up-regulation of the antigen, and not with in vitro selection of CD24⁺ cells. In tumors derived from subcutaneous injection of MA-11 mammospheres in athymic nude mice, 76.1 ± 9.7% of cells expressed CD24, vs. 0.5 ± 1% in MA-11 cells dissociated from mammospheres before injection. The tumorigenicity of sorted CD44⁺CD24⁻ and CD44⁺CD24^high MA-11 cells was equal. Single cell-sorted CD24⁻ and CD24^high MA-11 gave rise in vitro to cell populations with heterogeneous CD24 expression. Also, subcutaneous tumors derived from sorted CD24⁻ sub-populations and single-cell clones had levels of CD24 expression similar to the unsorted cells. To investigate whether the high expression of CD24 contributed to the tumorigenic potential of MA-11 cells, we silenced CD24 by shRNA. CD24 silencing (95%) resulted in no difference in tumorigenicity upon s.c. injection in athymic nude mice compared with mock-transduced MA-11 cells. Since CD24 silencing was maintained in vivo, our data suggest that the level of expression of CD24 is associated with but does not contribute to tumorigenicity. We then compared the molecular profile of the mammospheres with the adherent cell fraction. Gene expression profiling revealed that the increased tumorigenicity of MA-11 mammospheres was associated with changes in 10 signal transduction pathways, including MAP kinase, Notch and Wnt, and increased expression of aldehyde dehydrogenase, a cancer-initiating cell-associated marker. Our data demonstrate that (i) the level of CD24 expression is neither a stable feature of mammosphere-forming cells nor confers tumorigenic potential to MA-11 cells; (ii) cancer-initiating cell-enriched MA-11 mammospheres have activated specific signal transduction pathways, potential targets for anti-breast cancer therapy.     

Aberrant Lymphatic Endothelial Progenitors in Lymphatic Malformation Development

Lymphatic malformations (LMs) are vascular anomalies thought to arise from dysregulated lymphangiogenesis. These lesions impose a significant burden of disease on affected individuals. LM pathobiology is poorly understood, hindering the development of effective treatments. In the present studies, immunostaining of LM tissues revealed that endothelial cells lining aberrant lymphatic vessels and cells in the surrounding stroma expressed the stem cell marker, CD133, and the lymphatic endothelial protein, podoplanin. Isolated patient-derived CD133+ LM cells expressed stem cell genes (NANOG, Oct4), circulating endothelial cell precursor proteins (CD90, CD146, c-Kit, VEGFR-2), and lymphatic endothelial proteins (podoplanin, VEGFR-3). Consistent with a progenitor cell identity, CD133+ LM cells were multipotent and could be differentiated into fat, bone, smooth muscle, and lymphatic endothelial cells in vitro. CD133+ cells were compared to CD133− cells isolated from LM fluids. CD133− LM cells had lower expression of stem cell genes, but expressed circulating endothelial precursor proteins and high levels of lymphatic endothelial proteins, VE-cadherin, CD31, podoplanin, VEGFR-3 and Prox1. CD133− LM cells were not multipotent, consistent with a differentiated lymphatic endothelial cell phenotype. In a mouse xenograft model, CD133+ LM cells differentiated into lymphatic endothelial cells that formed irregularly dilated lymphatic channels, phenocopying human LMs. In vivo, CD133+ LM cells acquired expression of differentiated lymphatic endothelial cell proteins, podoplanin, LYVE1, Prox1, and VEGFR-3, comparable to expression found in LM patient tissues. Taken together, these data identify a novel LM progenitor cell population that differentiates to form the abnormal lymphatic structures characteristic of these lesions, recapitulating the human LM phenotype. This LM progenitor cell population may contribute to the clinically refractory behavior of LMs.     

Left ventricular heart aneurism--a new source of resident cardiac stem cells

In the past few years it has been established that the heart contains a reservoir of stem and progenitor cells that have the ability to differentiate in vitro and in vivo toward vascular and cardiac lineages and that show cardiac regeneration potential in vivo following injection into the infracted myocardium. The aim of the present study was to characterize cardiac stem cells in the tissue of chronic left ventricular aneurism. It was shown that human c-kit positive cells were scattered in fibrous, muscle and adipose parts of aneurism tissue. C-kit positive cells localized mainly in fibrous tissue nearby large vessels, however, c-kit positive cells did not express endothelial, smooth muscle or cardiomyocyte cell markers. Co-localization experiments demonstrated that all c-kit positive cells were of non-hematopoietic origin, since they did not express markers such as CD34 and CD45. Majority of c-kit positive cells expressed MDR1, but showed no proliferation activity (Ki67). It thus appears that aneurism tissue could be an alternative source of autologous cardiac stem cells. However, their regeneration capacity should be further explored.     

Heterogeneic nature of adult cardiac side population cells

Side population cells have been found in various types of adult tissue including heart and are presumed to be tissue-specific stem/progenitor cells. In the present study, we confirmed the presence of cardiac side population (cSP) cells, which showed both the Hoechst 33342 efflux ability and ABCG2 expression, in adult murine heart. Flow cytometric analysis showed that more than half of cSP cells expressed the endothelial marker VE-cadherin or the smooth muscle markers, α-smooth muscle actin and desmin. In addition, immunohistochemical analysis demonstrated that ABCG2⁺ cells were mainly localized within vascular walls. Quantitative RT-PCR analysis demonstrated that VE-cadherin⁻ cSP cells progressively expressed Nkx2.5 and cardiac troponin T with time in culture. VE-cadherin⁻ cSP cells also expressed mesodermal-mesenchymal-associated markers and differentiated into osteocytes and adipocytes. These results highlight the heterogeneic nature of cSP cells, consisting of vascular endothelial cells, smooth muscle cells, and mesenchymal stem/progenitor cells including potential cardiomyogenic cells.     

CD45RA−Foxp3 non-regulatory T cells in the CCR7−CD45RA−CD27+CD28+ effector memory subset are increased in synovial fluid from patients with rheumatoid arthritis

Increased numbers of regulatory T (Treg) cells are found in synovial fluid from patients with rheumatoid arthritis (RASF) compared with peripheral blood. However, Treg cells in RASF have been shown to have a decreased capacity to suppress T cells. Here we phenotypically classified CD4+ T cells in RASF into six subsets based on the expression of CD45RA, CCR7, CD27 and CD28, and demonstrated that the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in synovial fluid compared with peripheral blood. In addition, the proportion of Foxp3+ Treg cells in the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in RASF. Furthermore, most of the Foxp3+ Treg cells in RASF were non-suppressive CD45RA−Foxp3^low non-Treg cells, and the frequency of the non-Treg cells in the CCR7−CD45RA−CD27+CD28+ T_EM subset was significantly increased in RASF. Our findings suggest that the pro-inflammatory environment in RA joints may induce the increase of CD45RA−Foxp3^low non-Treg cells in synovial fluid.     

Sca-1+ cardiosphere-derived cells are enriched for Isl1-expressing cardiac precursors and improve cardiac function after myocardial injury

Background

Endogenous cardiac progenitor cells are a promising option for cell-therapy for myocardial infarction (MI). However, obtaining adequate numbers of cardiac progenitors after MI remains a challenge. Cardiospheres (CSs) have been proposed to have cardiac regenerative properties; however, their cellular composition and how they may be influenced by the tissue milieu remains unclear.

Methodology/Principal Finding

Using “middle aged” mice as CSs donors, we found that acute MI induced a dramatic increase in the number of CSs in a mouse model of MI, and this increase was attenuated back to baseline over time. We also observed that CSs from post-MI hearts engrafted in ischemic myocardium induced angiogenesis and restored cardiac function. To determine the role of Sca-1⁺CD45^- cells within CSs, we cloned these from single cell isolates. Expression of Islet-1 (Isl1) in Sca-1⁺CD45^- cells from CSs was 3-fold higher than in whole CSs. Cloned Sca-1⁺CD45^- cells had the ability to differentiate into cardiomyocytes, endothelial cells and smooth muscle cells in vitro. We also observed that cloned cells engrafted in ischemic myocardium induced angiogenesis, differentiated into endothelial and smooth muscle cells and improved cardiac function in post-MI hearts.

Conclusions/Significance

These studies demonstrate that cloned Sca-1⁺CD45^- cells derived from CSs from infarcted “middle aged” hearts are enriched for second heart field (i.e., Isl-1⁺) precursors that give rise to both myocardial and vascular tissues, and may be an appropriate source of progenitor cells for autologous cell-therapy post-MI.     

Endomyocardial biopsy derived adherent proliferating cells—A potential cell source for cardiac tissue engineering

Heart diseases are a leading cause of morbidity and mortality. Cardiac stem cells (CSC) are considered as candidates for cardiac‐directed cell therapies. However, clinical translation is hampered since their isolation and expansion is complex. We describe a population of human cardiac derived adherent proliferating (CAP) cells that can be reliably and efficiently isolated and expanded from endomyocardial biopsies (0.1 cm³). Growth kinetics revealed a mean cell doubling time of 49.9 h and a high number of 2.54 × 10⁷ cells in passage 3. Microarray analysis directed at investigating the gene expression profile of human CAP cells demonstrated the absence of the hematopoietic cell markers CD34 and CD45, and of CD90, which is expressed on mesenchymal stem cells (MSC) and fibroblasts. These data were confirmed by flow cytometry analysis. CAP cells could not be differentiated into adipocytes, osteoblasts, chondrocytes, or myoblasts, demonstrating the absence of multilineage potential. Moreover, despite the expression of heart muscle markers like α‐sarcomeric actin and cardiac myosin, CAP cells cannot be differentiated into cardiomyocytes. Regarding functionality, CAP cells were especially positive for many genes involved in angiogenesis like angiopoietin‐1, VEGF, KDR, and neuropilins. Globally, principal component and hierarchical clustering analysis and comparison with microarray data from many undifferentiated and differentiated reference cell types, revealed a unique identity of CAP cells. In conclusion, we have identified a unique cardiac tissue derived cell type that can be isolated and expanded from endomyocardial biopsies and which presents a potential cell source for cardiac repair. Results indicate that these cells rather support angiogenesis than cardiomyocyte differentiation. J. Cell. Biochem. 109: 564–575, 2010. © 2009 Wiley‐Liss, Inc.     

Human cardiac stem cells isolated from atrial appendages stably express c-kit

The in vivo studies of myocardial infarct using c-kit⁺/Lin⁻ cardiac stem cells (CSCs) are still in the early stage with margin or no beneficial effects for cardiac function. One of the potential reasons may be related to the absence of fully understanding the properties of these cells both in vitro and in vivo. In the present study, we aimed to systematically examine how CSCs adapted to in vitro cell processes and whether there is any cell contamination after long-term culture. Human CSCs were enzymatically isolated from the atrial appendages of patients. The fixed tissue sections, freshly isolated or cultured CSCs were then used for identification of c-kit⁺/Lin⁻ cells, detection of cell contamination, or differentiation of cardiac lineages. By specific antibody staining, we demonstrated that tissue sections from atrial appendages contained less than 0.036% c-kit⁺/Lin⁻ cells. For the first time, we noted that without magnetic activated cell sorting (MACS), the percentages of c-kit⁺/Lin⁻ cells gradually increased up to ∼40% during continuously culture between passage 2 to 8, but could not exceed >80% unless c-kit MACS was carried out. The resulting c-kit⁺/Lin⁻ cells were negative for CD34, CD45, CD133, and Lin markers, but positive for KDR and CD31 in few patients after c-kit MACS. Lin depletion seemed unnecessary for enrichment of c-kit⁺/Lin⁻ cell population. Following induced differentiation, c-kit⁺/Lin⁻ CSCs demonstrated strong differentiation towards cardiomyocytes but less towards smooth and endothelial cells. We concluded that by using an enzymatic dissociation method, a large number, or higher percentage, of relative pure human CSCs with stable expression of c-kit⁺ could be obtained from atrial appendage specimens within ∼4 weeks following c-kit MACS without Lin depletion. This simple but cost-effective approach can be used to obtain enough numbers of stably-expressed c-kit⁺/Lin⁻ cells for clinical trials in repairing myocardial infarction.     

Activation of myosin V-based motility and F-actin-dependent network formation of endoplasmic reticulum during mitosis

Putative myogenic and endothelial (myo-endothelial) cell progenitors were identified in the interstitial spaces of murine skeletal muscle by immunohistochemistry and immunoelectron microscopy using CD34 antigen. Enzymatically isolated cells were characterized by fluorescence-activated cell sorting on the basis of cell surface antigen expression, and were sorted as a CD34+ and CD45- fraction. Cells in this fraction were approximately 94% positive for Sca-1, and mostly negative (<3% positive) for CD14, 31, 49, 144, c-kit, and FLK-1. The CD34+/45- cells formed colonies in clonal cell cultures and colony-forming units displayed the potential to differentiate into adipocytes, endothelial, and myogenic cells. The CD34+/45- cells fully differentiated into vascular endothelial cells and skeletal muscle fibers in vivo after transplantation. Immediately after sorting, CD34+/45- cells expressed only c-met mRNA, and did not express any other myogenic cell-related markers such as MyoD, myf-5, myf-6, myogenin, M-cadherin, Pax-3, and Pax-7. However, after 3 d of culture, these cells expressed mRNA for all myogenic markers. CD34+/45- cells were distinct from satellite cells, as they expressed Bcrp1/ABCG2 gene mRNA (Zhou et al., 2001). These findings suggest that myo-endothelial progenitors reside in the interstitial spaces of mammalian skeletal muscles, and that they can potentially contribute to postnatal skeletal muscle growth.     

Endocardial Tip Cells in the Human Embryo – Facts and Hypotheses

Experimental studies regarding coronary embryogenesis suggest that the endocardium is a source of endothelial cells for the myocardial networks. As this was not previously documented in human embryos, we aimed to study whether or not endothelial tip cells could be correlated with endocardial-dependent mechanisms of sprouting angiogenesis. Six human embryos (43–56 days) were obtained and processed in accordance with ethical regulations; immunohistochemistry was performed for CD105 (endoglin), CD31, CD34, α-smooth muscle actin, desmin and vimentin antibodies. Primitive main vessels were found deriving from both the sinus venosus and aorta, and were sought to be the primordia of the venous and arterial ends of cardiac microcirculation. Subepicardial vessels were found branching into the outer ventricular myocardium, with a pattern of recruiting α-SMA+/desmin+ vascular smooth muscle cells and pericytes. Endothelial sprouts were guided by CD31+/CD34+/CD105+/vimentin+ endothelial tip cells. Within the inner myocardium, we found endothelial networks rooted from endocardium, guided by filopodia-projecting CD31+/CD34+/CD105+/ vimentin+ endocardial tip cells. The myocardial microcirculatory bed in the atria was mostly originated from endocardium, as well. Nevertheless, endocardial tip cells were also found in cardiac cushions, but they were not related to cushion endothelial networks. A general anatomical pattern of cardiac microvascular embryogenesis was thus hypothesized; the arterial and venous ends being linked, respectively, to the aorta and sinus venosus. Further elongation of the vessels may be related to the epicardium and subepicardial stroma and the intramyocardial network, depending on either endothelial and endocardial filopodia-guided tip cells in ventricles, or mostly on endocardium, in atria.     

Characterization of Discrete Subpopulations of Progenitor Cells in Traumatic Human Extremity Wounds

Here we show that distinct subpopulations of cells exist within traumatic human extremity wounds, each having the ability to differentiate into multiple cells types in vitro. A crude cell suspension derived from traumatized muscle was positively sorted for CD29, CD31, CD34, CD56 or CD91. The cell suspension was also simultaneously negatively sorted for either CD45 or CD117 to exclude hematopoietic stem cells. These subpopulations varied in terms their total numbers and their abilities to grow, migrate, differentiate and secrete cytokines. While all five subpopulations demonstrated equal abilities to undergo osteogenesis, they were distinct in their ability to undergo adipogenesis and vascular endotheliogenesis. The most abundant subpopulations were CD29+ and CD34+, which overlapped significantly. The CD29+ and CD34+ cells had the greatest proliferative and migratory capacity while the CD56+ subpopulation produced the highest amounts of TGFß1 and TGFß2. When cultured under endothelial differentiation conditions the CD29+ and CD34+ cells expressed VE-cadherin, Tie2 and CD31, all markers of endothelial cells. These data indicate that while there are multiple cell types within traumatized muscle that have osteogenic differentiation capacity and may contribute to bone formation in post-traumatic heterotopic ossification (HO), the major contributory cell types are CD29+ and CD34+, which demonstrate endothelial progenitor cell characteristics.     

Resident cardiac immune cells and expression of the ectonucleotidase enzymes CD39 and CD73 after ischemic injury

Background

The ectoenzymes CD39 and CD73 are expressed by a broad range of immune cells and promote the extracellular degradation of nucleotides to anti-inflammatory adenosine. This study explored the abundance of CD73 and CD39 on circulating and resident cardiac leukocytes and coronary endothelial cells under control conditions and in response to inflammation following myocardial ischemia and reperfusion (I/R).

Methods and Results

A method was elaborated to permit FACS analysis of non-myocardial cells (resident leukocytes, coronary endothelium and CD31⁻ CD45⁻ cells) of the unstressed heart. Under control conditions the murine heart contained 2.3×10³ resident leukocytes/mg tissue, the most prominent fraction being antigen-presenting mononuclear cells (CD11b⁺ CD11c⁺ F4/80⁺ MHCII⁺) followed by B-cells, monocytes and T-cells. CD73 was highly expressed on circulating and resident cardiac lymphoid cells with little expression on myeloid cells, while the opposite was true for CD39. Cardiomyocytes and erythrocytes do not measurably express CD39/CD73 and CD39 dominates on coronary endothelium. Three days after I/R, CD73 was significantly upregulated on invading granulocytes (2.8-fold) and T-cells (1.5-fold). Compared with coronary endothelial cells, CD73 associated with leukocytes comprised 2/3 of the total cardiac CD73.

Conclusion

Our study suggests that extracellular ATP formed during I/R is preferentially degraded by CD39 present on myeloid cells, while the formation of immunosuppressive adenosine is mainly catalysed by CD73 present on granulocytes and lymphoid cells. Upregulated CD73 on granulocytes and T-cells infiltrating the injured heart is consistent with the existence of an autocrine adenosinergic loop which may promote the healing process.     

Kinetic mechanism of the Ca2+-dependent switch-on and switch-off of cardiac troponin in myofibrils

The kinetics of Ca²⁺-dependent conformational changes of human cardiac troponin (cTn) were studied on isolated cTn and within the sarcomeric environment of myofibrils. Human cTnC was selectively labeled on cysteine 84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole and reconstituted with cTnI and cTnT to the cTn complex, which was incorporated into guinea pig cardiac myofibrils. These exchanged myofibrils, or the isolated cTn, were rapidly mixed in a stopped-flow apparatus with different [Ca²⁺] or the Ca²⁺-buffer 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid to determine the kinetics of the switch-on or switch-off, respectively, of cTn. Activation of myofibrils with high [Ca²⁺] (pCa 4.6) induced a biphasic fluorescence increase with rate constants of >2000 s⁻¹ and ∼330 s⁻¹, respectively. At low [Ca²⁺] (pCa 6.6), the slower rate was reduced to ∼25 s⁻¹, but was still ∼50-fold higher than the rate constant of Ca²⁺-induced myofibrillar force development measured in a mechanical setup. Decreasing [Ca²⁺] from pCa 5.0-7.9 induced a fluorescence decay with a rate constant of 39 s⁻¹, which was approximately fivefold faster than force relaxation. Modeling the data indicates two sequentially coupled conformational changes of cTnC in myofibrils: 1), rapid Ca²⁺-binding (k_B ≈ 120 μM⁻¹ s⁻¹) and dissociation (k_D ≈ 550 s⁻¹); and 2), slower switch-on (k_on = 390s⁻¹) and switch-off (k_off = 36s⁻¹) kinetics. At high [Ca²⁺], ∼90% of cTnC is switched on. Both switch-on and switch-off kinetics of incorporated cTn were around fourfold faster than those of isolated cTn. In conclusion, the switch kinetics of cTn are sensitively changed by its structural integration in the sarcomere and directly rate-limit neither cardiac myofibrillar contraction nor relaxation.     

Fetal hepatic expression of 5-lipoxygenase activating protein is confined to colonizing hematopoietic cells

Leukotriene C₄ is a potent inflammatory mediator formed from arachidonic acid and glutathione. 5-Lipoxygenase (5-LO), 5-lipoxygenase activating protein (FLAP) and leukotriene C₄ synthase (LTC₄S) participate in its biosynthesis. We report evidence from insitu hybridization experiments that FLAP mRNA is abundantly expressed in fetal mouse liver from e11.5 until delivery. In contrast very little or no FLAP mRNA was detected in adult liver. The fetal expression in liver was not uniform but occurred in patches. Cells from e15.5 livers were fractionated by fluorescence activated cell sorting into hepatocytes and other CD45⁻ cells and CD45⁺ hematopoietic cells. The latter were further separated into immature (Lin⁻) and mature (Lin⁺) cells and analyzed for FLAP mRNA content by quantitative RT-PCR. FLAP mRNA expression was confined to CD45⁺ cells and the mature cells had approximately 4-fold higher FLAP mRNA levels compared to the immature cells.