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Anti-DKK1 Enhances the Early Osteogenic Differentiation of Human Adipose-Derived Stem/Stromal Cells
Get PDF: Adipose-derived stem/stromal cells (ASCs) have been previously used for bone repair. However, significant cell heterogeneity exists within the ASC population, which has the potential to result in unreliable bone tissue formation and/or low efficacy. Although the use of cell sorting to lower cell heterogeneity is one method to improve bone formation, this is a technically sophisticated and costly process. In this study, we tried to find a simpler and more deployable solution-blocking antiosteogenic molecule Dickkopf-1 (DKK1) to improve osteogenic differentiation. Human adipose-derived stem cells were derived from = 5 samples of human lipoaspirate. In vitro, anti-DKK1 treatment, but not anti-sclerostin (SOST), promoted ASC osteogenic differentiation, assessed by alizarin red staining and real-time polymerase chain reaction (qPCR). Increased canonical Wnt signaling was confirmed after anti-DKK1 treatment. Expression levels of DKK1 peaked during early osteogenic differentiation (day 3). Concordantly, anti-DKK1 supplemented early (day 3 or before), but not later (day 7) during osteogenic differentiation positively regulated osteoblast formation. Finally, anti-DKK1 led to increased transcript abundance of the Wnt inhibitor SOST, potentially representing a compensatory cellular mechanism. In sum, DKK1 represents a targetable "molecular brake" on the osteogenic differentiation of human ASC. Moreover, release of this brake by neutralizing anti-DKK1 antibody treatment at least partially rescues the poor bone-forming efficacy of ASC
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Identifying human perivascular stem cell subsets
Get PDFPerivascular stem cells (PSCs) include pericytes and adventitial cells. PSCs present
multiple properties. PSCs are involved in angiogenesis, immunoregulation, and
haematopoiesis support and are multi-lineage progenitor cells. Therefore, PSCs are a
heterogeneous group of cells. We investigated PSC subsets based on novel markers:
CD10 and CD107a. We analysed whether the expression of CD10 or CD107a on
PSCs from foetal muscle correlates with the capability of differentiation and
fibroblast-colony forming unit (CFU-f) content. The CD10-positive or CD107apositive
PSCs were separated from CD10-negative or CD107a-negative PSCs by cell
sorting. CFU-f was quantified. The differentiation of PSC subsets in culture was
documented by cytochemistry. We confirmed that CD10 and CD107a PSCs subsets
exist in multiple human tissues. CD10-positive and CD10-negative PSC subsets
show similar ability for both CFU-f potential and osteogenesis in vitro.CD107anegative
cells show higher CFU-f potential. However, CD107a-positive PSCs were
associated with a higher osteogenic differentiation potential in human foetal muscle
in vitro. Our study provides early evidence that CD107a-positive adventitial cells
present a subset that is prone to differentiate into osteoblasts
Mesenchymal progenitor cells in human dental pulp: purification and characterisation
Get PDFINTRODUCTION:
Characterising the dental pulp perivascular cells will allow understanding whether these blood vessel associated cells have tissue regenerative potential that can be used for craniofacial regeneration, including bone, nerve, and tooth repair. Perivascular cells (pericytes and adventitial cells) can be purified from multiple human organs including placenta, bone marrow, pancreas, fat and muscle, and exhibit in culture and in vivo multilineage mesodermal developmental potential, implying the perivascular origin of the elusive, culture-derived mesenchymal stem cells (MSCs). Perivascular cells are ubiquitous in the organism and their developmental potential is partially imprinted by their tissue of origin. Pericytes in the dental pulp, which are deriving from primary or permanent teeth, originate from migrating cranial neural crest cells, which, theoretically, could exhibit superior potential to regenerate neural tissue. To test this, my aim was to characterise dental pulp cells and to evaluate their regenerative capacity.
MATERIAL AND METHODS:
To achieve this goal, we identified and isolated perivascular cells present in the human dental pulp. Dental pulp tissue was isolated from healthy third molar teeth from adults younger than 30 years old. Firstly, immunohistochemistry was performed to determine the presence of cell surface markers on dental pulp pericytes and adventitial cells using markers such as αSMA, PDGFRβ, LepR, CD107a, CD146, NG2 and CD34. Blood vessels were next characterised using MSC markers such as CD44, CD90, and CD29 and endothelial cell markers CD54, CD34, UEA-1, and vWF on pulp sections by immunofluorescence microscopy. Secondly, purification of dental pulp perivascular cells was performed using flow cytometry, followed by culturing and differentiation assays towards osteogenic, adipogenic and neurogenic cell lineages.
RESULTS:
We found that the phenotype of pericytes in the dental pulp in vivo is similar to that of pericytes in other human organs previously documented. They express CD146, ALP and PDGFRβ and lack endothelial cell markers such as CD34, vWF, CD54 or UEA-1. Importantly, we also found that pericytes are heterogeneous and can be further discriminated based on NG2, αSMA and LepR that coincide with a particular blood vessel type or blood vessel diameter. For example, αSMA is mainly expressed in microvascular pericytes and occasionally in capillaries, similar to LepR. NG2 is not expressed in venules but it is expressed in capillaries and arterioles. In contrast, MSC markers CD90, CD44, and CD29 were expressed on all pericytes suggesting that MSCs reside in heterogeneous perivascular niches.
Dental pulp mesenchymal stromal cells were next derived from CD146+CD34-CD56-CD45- pericytes or NG2+ and NG2- pericyte subsets and from CD146-CD34+CD56-CD45- adventitial cells, another perivascular cell source of MSCs located around large vessels. Our data show that, similar to MSCs, all perivascular cell subsets we purified were able to adhere to plastic dish, to expand in culture, and to efficiently accumulate calcium deposits upon osteogenic induction, reminiscent of bone development. However, most of cell lines tested were not adipogenic with the exception of rare cells in the NG2+ pericytes subset. Indeed, lipid droplets were consistently found accumulating only in these purified pericytes from 14 days onwards, albeit at a low frequency.
Finally, we discovered that, in situ, neural cells, positive for CD56 and vimentin, share markers with pericytes such as NG2 and CD146. We also found that in vitro, our perivascular cell-derived mesenchymal stromal cell lines express tubulin β III upon neuronal induction although these results require further confirmation.
CONCLUSIONS:
We documented the molecular signature of pericytes in the human dental pulp. We demonstrated that perivascular cell subsets can be prospectively purified to homogeneity and that perivascular cell-derived stromal cell cultures can be established in vitro. Intriguingly, our in vitro MSC functional assays showed that these cells are not multipotent, and thus are not genuine MSCs. They are, in contrast, very potent bone progenitors with a low frequency of arterial pericytes being adipogenic. Importantly, we here showed a marker overlap between neural cells and pericytes. Whether these cell types are developmentally linked remains to be established
Role of PDGF-B/PDGFR: signalling in definitive haematopoiesis
Get PDFThe first haematopoietic stem cells (HSCs) are generated in the dorsal aorta (DA) of the midgestation mouse embryo. Signals from the microenvironment are required for HSC generation. However, the signals and identity of cells releasing them in vivo remain unknown. In the present work, we identified at least three populations of perivascular cells surrounding the DA based on the expression of the perivascular cell markers NG2, PDGFRβ and αSMA. NG2+ PDGFRβ+ αSMA+ pericytes/vascular smooth muscle cells (PCs/vSMCs) and NG2- PDGFRβ+ αSMA- sub-pericytes (Sub-PCs) were found to be enriched in HSC-supportive genes described in the adult bone marrow (BM). As both populations express PDGFRβ, which has recently been shown to mediate HSC specification in zebrafish, and PDGF-B/PDGFRβ signalling is also required for the recruitment of pericytes to the developing blood vessel wall, we hypothesised that PDGF-B/PDGFRβ signalling is required to generate the first HSCs in vivo. To answer this question, we used PDGFRβ knock-out (KO) and PDGF-Bret KO mice, both of which have a defective pericyte recruitment to blood vessels and defective or absent PDGF-B/PDGFRβ signalling. Results from our haematopoietic progenitor assays (CFU-Cs) and transplantations show that the germline deletion of PDGFRβ affects both haematopoietic progenitor numbers and HSC activity in the E11 AGM. HSPCs in other haematopoietic organs are not affected at this stage. PDGF-Bret KO mice showed no defects in midgestation HSPCs, but AGM HSCs failed to reconstitute secondary recipients. Together, these data suggest that PDGFB/PDGFRβ signalling is required for AGM haematopoiesis. We found that perivascular stromal cells surrounding the DA are not affected by these mutations, nor the integrity of the blood vessel, suggesting that PDGFB/PDGFRβ signalling is not required for PC/vSMCs recruitment to the DA. We therefore hypothesised that PDGF-B/PDGFRβ signalling is either required in the niche for HSC specification and/or generation, and/or that PDGFRβ+ cells are precursors of HSCs. Tracing experiments using PDGFRβ-Cre;TdTomato mice found that a subset of AGM HSPCs derive from PDGFRβ-Cre precursors and that both Tomato- and Tomato+ E14 foetal liver (FL) and BM cells
reconstitute irradiated recipients. Together these data suggest that adult HSCs have distinct developmental origins, one of them deriving from PDGFRβ+ cells. In conclusion, our results define PDGFRβ signalling as a key component of the HSC generating niche in the mouse embryo, and that a subset of HSCs derive from PDGFRβ+ precursors
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Stem cells and their “niche” in the human heart: toward cell therapies of cardiac disease
No full textReplacement of dead cardiomyocytes by scar tissue, unable to mediate normal cardiac
contraction, is the most dramatic change following heart infarction. Current dogma
investigates potential endogenous “self-repairing” capacity of the myocardium as a means to
minimize such degenerative process. Spontaneous regeneration of the myocardium could be
exploited to treat severe and frequently fatal conditions. Post ischaemic intrinsic cardiac
healing capability could arise because: i) Cardiomyocytes may proliferate and regenerate
damaged tissue, or; i) Endogenous cardiac stem cells (CSCs) may divide and differentiate to
exhibit cardiac repairing potential. Although CSCs existence has been documented, their
identity, intra-cardiac localization and exact potential remain elusive. Cardiac pericytes, which
ensheath blood vessels and express cardiac repair capabilities, could interact with c-kit+ CSCs
nested in vascular niches during cellular response to injury. I tested the hypothesis that
pericytes and endogenous cardiac progenitor’s stem cells might increase their interaction
within vascular niches under ischaemic conditions.
Focussing on the potential of cell therapies of cardiac disease, I investigated the spatial
relationship between pericytes and endogenous cardiac progenitors within stem cells' niches
localised in different regions of the human foetal, adult healthy and ischaemic heart.
Immunostaining of foetal human cardiac tissues showed that c-kit+ cells expression and their
association with pericytes decrease with heart development.
Clear decrease is already evident
by 19th week of the gestation. Pericytes and c-kit+ cell populations isolated from foetal hearts
and expanded in culture reveal that pericytes’ cells express higher levels of the mesodermal
cardiac progenitor factor KDR: 3751± 61(SD) vs 398± 19.9 (SD); (P< 0.05) and for the marker
towards cardiac lineage Islet1: 1146±155 (SD) vs 728± 124 (SD); (P< 0.05) while c-kit+ cells
express higher levels than pericytes of the stemness marker SSEA3:1655± 40.6 (SD) vs 747±
27 (SD); (P< 0.05), known to progressively decrease with cell differentiation. Absence of
staining for CD31 marker in cultured cardiac pericytes and c-kit+ cells is replicated by results
of endothelial differentiation assessment, which shows that cardiac pericytes and c-kit+ cells
do not form CD31+ networks. The cardiac marker α-actin was present in both cell populations.
In healthy adult heart, pericytes marker CD146 localise within the vasculature. Following
ischaemia this pericyte marker becomes also evident outside the vasculature. In healthy adult
atrium, c-kit expression is low and coexpression with other markers inconspicuous. Ischaemia
leads to increased c-kit expression in the microvasculature.
Furthermore, following ischaemia
c-kit, endothelium and pericyte markers colocalize within the same atrial cells. Colocalization
studies in ischaemic hearts revealed low levels of co-occurrence (M1/M2) between c-kit and
vascular markers in vessels <50μm but a high degree of correlation (PCC). Ischaemia leads to
increased c-kit expression, particularly in blood vessels <50um diameter. Blood
vessels >50μm diameter show mostly, staining for endothelial (vWF) and pericyte (CD146)
markers. Acute ischaemia of the left ventricle affected the detection of cardiac stem cells
markers in the infarcted area.
The absence of coexpression of markers during acute ischaemia of the left ventricle suggests
that post-ischaemia markers coexpresion is time dependent.
CONCLUSION:
Foetal heart pericytes and c-kit+/CD117 cells express early cardiac
transcription factors and show trans-differentiation potential, which decreases in healthy adult
hearts. The preservation and activity of cardiac stem cells’ niches within the atrium
vasculature, appears re-activated in post-ischaemic hearts.
Better understanding of cardiac c-kit+
and pericyte cells’ interactions during-human embryonic development and during
ischaemia may identify alternative novel therapeutic strategy against coronary artery disease
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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