BACKGROUND

Recent findings on human hematopoietic stem cell (HSC) properties suggest a possible therapeutic role of human umbilical cord blood (UCB) HSC-based cellular therapies in the treatment of myocardial infarction.

METHODS

Nine UCB units were subjected to sequential red cell removal, freezing, and postthawing CD133+ HSC immunoselection by a clinical-grade, CE-approved, magnetic apparatus and microbead-coated anti-CD133 monoclonal antibody. Selected UCB CD133+ cells were cultured in vitro in medium supporting either endothelial or cardiomyocytic differentiation in parallel experiments.

RESULTS

Immunoselection allowed recovery of 79 percent of initial CD133+ cells with a CD133+ cell purity of 81 percent, on average. Parallel cultures showed the appearance of endothelial markers (VE-cadherin, CD146, and KDR and bright expression of CD105), morphofunctional features of endothelium in endothelial-supporting cultures, of cardiac muscle proteins (troponin I and myosin ventricular heavy chain alpha/beta; MYHC) and specific gene expression (GATA4, NKX2.5, troponin I, and MYHC) in cardiomyocyte-oriented cultures.

CONCLUSIONS

The appearance of both endothelial- and cardiomyocyte-like cells from parallel cultures of frozen-thawed-immunoselected UCB CD133+ cells by a clinical-grade method and previously reported data on lack of major signs of rejection of these cells in immunocompetent rats subjected to experimental liver damage suggest a possible role of these allogeneic HSCs in cell therapies designed for regenerative treatments of ischemic diseases of human myocardium.

CD146 is a newly identified endothelial biomarker that has been implicated in angiogenesis. Though in vitro angiogenic function of CD146 has been extensively reported, in vivo evidence is still lacking. To address this issue, we generated endothelial-specific CD146 knockout (CD146(EC-KO)) mice using the Tg(Tek-cre) system. Surprisingly, these mice did not exhibit any apparent morphological defects in the development of normal retinal vasculature. To evaluate the role of CD146 in pathological angiogenesis, a xenograft tumor model was used. We found that both tumor volume and vascular density were significantly lower in CD146(EC-KO) mice when compared to WT littermates. Additionally, the ability for sprouting, migration and tube formation in response to VEGF treatment was impaired in endothelial cells (ECs) of CD146(EC-KO) mice. Mechanistic studies further confirmed that VEGF-induced VEGFR-2 phosphorylation and AKT/p38 MAPKs/NF-κB activation were inhibited in these CD146-null ECs, which might present the underlying cause for the observed inhibition of tumor angiogenesis in CD146(EC-KO) mice. These results suggest that CD146 plays a redundant role in physiological angiogenic processes, but becomes essential during pathological angiogenesis as observed in tumorigenesis.

BACKGROUND

The inflammatory properties of vein endothelium in relation to chronic venous disease (CVD) have been poorly investigated. Therefore, new insights on the characteristics of large vein endothelium would increase our knowledge of large vessel physiopathology.

RESULTS

Surgical specimens of veins were obtained from the tertiary venous network (R3) and/or saphenous vein (SF) of patients affected by CVD and from control individuals. Highly purified venous endothelial cell (VEC) cultures obtained from CVD patients were characterized for morphological, phenotypic and functional properties compared to control VEC. An increase of CD31/PECAM-1, CD146 and ICAM-1 surface levels was documented at flow cytometry in pathological VEC with respect to normal controls. Of note, the strongest expression of these pro-inflammatory markers was observed in VEC obtained from patients with more advanced disease. Similarly, spontaneous cell proliferation and resistance to starvation was higher in pathological than in normal VEC, while the migratory response of VEC showed an opposite trend, being significantly lower in VEC obtained from pathological specimens. In addition, in keeping with a higher baseline transcriptional activity of NF-kB, the release of the pro-inflammatory cytokines osteoprotegerin (OPG) and vascular endothelial growth factor (VEGF) was higher in pathological VEC cultures with respect to control VEC. Interestingly, there was a systemic correlation to these in vitro data, as demonstrated by higher serum OPG and VEGF levels in CVD patients with respect to normal healthy controls.

CONCLUSIONS

Taken together, these data indicate that large vein endothelial cells obtained from CVD patients exhibit a pro-inflammatory phenotype, which might significantly contribute to systemic inflammation in CVD patients.

Circulating endothelial cells (CEC) and endothelial microparticles (EMP) are emerging as markers of endothelial repair and activation/apoptosis. Although significant changes in the number of CEC and EMP in pathological conditions have been reported, their reliable identification and quantification still remain a technical challenge. Here, we present a novel methodology for the identification and quantitation of CEC and EMP based on multicolor flow cytometry. Using a lyse/no wash protocol, we observed that in 50 μl of peripheral blood, the large majority of events expressing an endothelial phenotype (CD45-/CD146+/CD34+) are due to non-nucleated particles (DRAQ5-) carrying mitochondrial activity (MitoTracker+) and, therefore, classified as EMP. We enumerated circulating EMP by single platform absolute count in a lyse/no wash four-color flow-cytometric procedure, which allowed the distinction, within the whole endothelial compartment, of EMP derived from endothelial progenitors (CD45-/CD146+/CD34+/CD117+) and from mature endothelial cells (CD45-/CD146+/CD34+/CD117-). A significant increase in both subsets was observed in patients with diabetes mellitus. Thus, this simple and highly reproducible method may be useful for monitoring endothelial dysfunction in clinical settings.

Endothelial cell (EC) dysfunction is a key feature of multiple organ injury, the primary cause of fatality seen in critically ill patients. Although the development of EC dysfunction in the heart and lung is well studied in sepsis, it remains unclear in the liver. Herein, we report that liver sinusoidal ECs (LSECs; defined as CD146(+)CD45(-)) exhibit increased intercellular adhesion molecule-1 (CD54) and Fas in response to sepsis induced by cecal ligation and puncture (CLP). By using magnetically enriched LSEC (CD146(+)) populations, we show evidence of marked apoptosis, with a twofold decline in viable LSECs in CLP animals compared with sham controls. These changes and increased serum alanine aminotransferase levels were all mitigated in septic Fas(-/-) and Fas ligand(-/-) animals. Although we previously reported increased numbers of Fas ligand expressing CD8(+) T lymphocytes in the septic liver, CD8(+) T-cell deficiency did not reverse the onset of LSEC apoptosis/damage. However, Kupffer cell depletion with clodronate liposomes resulted in greater apoptosis and Fas expression after CLP and a decrease in glycoprotein 130 expression on LSECs, suggesting that STAT3 activation may protect these cells from injury. Our results document a critical role for death receptor-mediated LSEC injury and show the first evidence that Kupffer cells are essential to the viability of LSECs, which appears to be mediated through glycoprotein 130 expression in sepsis.

Calcium hydroxide has been extensively and steadily used for direct pulp capping in modern clinical dentistry. As it was known to have potential to induce hard tissue repair, this chemical has been applied to the exposed dental pulp and the hard tissue is expected to be regenerated above the pulp. During the reparative process of exposed pulp, primary odontoblasts that were lost as a result of extensive damage are replaced with newly differentiated odontoblast-like cells. This process is known to follow the sequential steps of proliferation, migration, and differentiation of progenitor cells. This research will examine the relationship between calcium hydroxide and the recruitment, proliferation, and mineralization of postnatal dental stem cells, obtained from an immature dental tissue of beagle dogs. Immunocytochemical staining and reverse transcriptase-polymerase chain reaction were used to identify the putative stem cell markers. Immunoblot analysis, wound healing assay, cell migration assay, and alizarin red staining were used to evaluate proliferation, migration, and mineralization capacity of the calcium hydroxide-treated stem cells. As an in vivo study, a combination of calcium hydroxide and autologous dental pulp stem cells (DPSCs) was applied for the treatment of intentionally created tooth defects on the premolars and the molars in beagle dogs to observe dentin regeneration. Ex vivo expanded DPSCs and periodontal ligament stem cells expressed STRO-1 and CD146, the mesenchymal stem cell markers. It was evident that calcium hydroxide increased recruitment, migration, proliferation, and mineralization of the DPSCs and periodontal ligament stem cells. Such results are valuable for future availability of DPSCs, which are recently focused as the stem cell reservoir for regeneration of dentin upon tooth injury, as well as for elucidation of the role of calcium hydroxide in pulp capping therapy.

OBJECTIVE

Stem Cells residing in the Apical Papilla (SCAP) of human permanent teeth represent a promising cell source for dental tissue regeneration. Therefore, the functional and molecular properties of specific subpopulations existing within heterogeneous cultures should be further investigated to give insight whether their selection could be beneficial for targeted therapeutic applications.

METHODS

In this study we extensively characterized SCAP cultures established from 10 healthy subjects, as well as their STRO-1(pos/)CD146(pos) and STRO-1(neg/)CD146(pos) subpopulations isolated with fluorescence-activated cell sorting. SCAP were analyzed for embryonic (Nanog, Oct3/4, SSEA-3, TRA-1-60), mesenchymal (STRO-1, CD146/MUC18, CD105/endoglin, CD24, CD90/Thy-1, CD81-TAPA, CD34, CD49f/a6-integrin), neural (CD271/NGFR, nestin) and hematopoietic (CD117/c-kit, CD45) stem cell (SC) markers using flow cytometry. Multipotentiality was evaluated with culture specific staining (Alizarin-Red-S, Oil- Red-O) and RT-PCR analysis for osteo/odontogenic (DSPP, BSP, ALP, osteocalcin, osteonectin, BMP-2, Runx2), adipogenic (lipoprotein-lipase-LPL) and neurogenic (Neurofilament/NFL-L, nestin, β-tubulin-III, NCAM) markers.

RESULTS

Our results showed that the STRO-1(pos)/CD146(pos) subpopulation demonstrated higher CFU efficiency and much higher expression of several embryonic and mesenchymal SC markers compared to the non-sorted SCAP. They also showed enhanced odontogenic differentiation potential, as evidenced by higher mineralization capacity and expression of osteo/odontogenic markers. By contrast, absence of STRO-1 in the STRO-1(neg)/CD146(pos) subpopulation yielded the opposite results and was associated with significant downgrading of the above-mentioned properties.

CONCLUSIONS

These results suggest that STRO-1(pos)/CD146(pos) SCAP cells represent a very promising adult MSCs source with enhanced multipotent SC properties that could be easily isolated with simple flow cytometric methods to be used for tissue engineering applications.

Human endometrium regenerates on a cyclical basis each month, likely mediated by endometrial stem/progenitor cells. Several types of stem/progenitor cells have been identified: CD140b+CD146+ or SUSD2+ endometrial mesenchymal stem cells (eMSCs), N-cadherin+ endometrial epithelial progenitor cells (eEPs), and side population (SP) cells, a heterogeneous population predominantly comprising endothelial cells. eMSCs reside in a perivascular niche and likely mediate angiogenesis and stromal regeneration. Human eEPs are located in the bases of glands in the basalis and are likely more primitive than SSEA-1+ basalis epithelial cells. Endometrial stem/progenitor cells may contribute to the pathogenesis of endometriosis by their retrograde shedding into the pelvic cavity, either after menarche or as a result of neonatal uterine bleeding. eMSCs may have a role in the generation of progesterone-resistant phenotype of endometrial stromal fibroblasts (eSFs) in endometriosis. In future clinical practice, endometrial stem/progenitor cells may be used to establish diagnosis of endometriosis or as therapeutic targets.

Regenerative endodontic procedures are stem cell-based treatments for immature teeth with pulp necrosis. The translation of regenerative endodontic procedures into treating mature teeth depends, among other factors, on the availability and delivery of mesenchymal stem cells (MSCs) into the root canal system. The aim of this clinical study was to evaluate whether evoked bleeding from the periapical tissues elicits the influx of MSCs into the root canal system in mature teeth with apical lesions. Participants included in this study (N = 20) were referred for endodontic treatment of mature teeth with apical lesions. Following chemomechanical debridement, intracanal bleeding from the periapical tissues was achieved, and intracanal blood samples were collected. A positive blood aspirate was also collected in the cartridges during local anesthesia. Total RNA was isolated and used as a template in quantitative reverse transcription polymerase chain reactions using MSC-specific arrays. Data were analyzed with the Wilcoxon signed-rank test, and correlation between gene expression and sex or age was tested with Spearman's rank correlation coefficient test. In addition, MSCs were isolated from an intracanal bleeding sample and subjected to flow cytometry and quantitative osteogenesis assay. Last, the presence and distribution of MSCs within periradicular lesions were evaluated with immunohistochemistry (n = 4). The MSC markers CD73, CD90, CD105, and CD146 were significantly upregulated, with median fold change values of 2.9, 31.7, 4.6, and 6.8, respectively. Conversely, the negative marker for MSCs, CD45, was significantly downregulated (median, -2.7). There was no correlation with age, sex, tooth type, or treatment for any of the evaluated genes. Isolated intracanal cells coexpressed MSC markers and demonstrated robust mineralizing differentiation potential. Finally, immunohistochemical analysis revealed that MSCs were found compartmentalized mainly within vasculature structures located in periapical lesions. Collectively, findings indicate that the evoked-bleeding technique delivers MSCs into the root canal system in mature teeth with apical lesions.

Heterogeneity of mesenchymal stem cells (MSCs) influences the cell therapy outcome and the application in tissue engineering. Also, the application of subpopulations of MSCs in cartilage regeneration remains poorly characterized. CD146+ MSCs are identified as the natural ancestors of MSCs and the expression of CD146 are indicative of greater pluripotency and self-renewal potential. Here, we sorted a CD146⁺ subpopulation from adipose-derived mesenchymal stem cells (ADSCs) for cartilage regeneration. Methods: CD146⁺ ADSCs were sorted using magnetic activated cell sorting (MACS). Cell surface markers, viability, apoptosis and proliferation were evaluated in vitro. The molecular signatures were analyzed by mRNA and protein expression profiling. By intra-articular injections of cells in a rat osteochondral defect model, we assessed the role of the specific subpopulation in cartilage microenvironment. Finally, CD146⁺ ADSCs were combined with articular cartilage extracellular matrix (ACECM) scaffold for long term (3, 6 months) cartilage repair. Results: The enriched CD146⁺ ADSCs showed a high expression of stem cell and pericyte markers, good viability, and immune characteristics to avoid allogeneic rejection. Gene and protein expression profiles revealed that the CD146⁺ ADSCs had different cellular functions especially in regulation inflammation. In a rat model, CD146⁺ ADSCs showed a better inflammation-modulating property in the early stage of intra-articular injections. Importantly, CD146⁺ ADSCs exhibited good biocompatibility with the ACECM scaffold and the CD146⁺ cell-scaffold composites produced less subcutaneous inflammation. The combination of CD146⁺ ADSCs with ACECM scaffold can promote better cartilage regeneration in the long term. Conclusion: Our data elucidated the function of the CD146⁺ ADSC subpopulation, established their role in promoting cartilage repair, and highlighted the significance of cell subpopulations as a novel therapeutic for cartilage regeneration.

Perivascular stem cells (PSCs) are the natural ancestors of mesenchymal stem cells (MSCs) and are the stem cells responsible for homeostasis and repair in vivo. Prospectively identified and isolated PSCs have demonstrated increased plasticity and osteogenic potential. Cells from the infrapatellar fat pad (IFP) have demonstrated increased chondrogenic potential compared with those from subcutaneous fat. This research assessed the chondrogenic potential of IFP PSCs compared with MSCs from the IFP and bone marrow. Immunohistochemistry demonstrated the location of perivascular markers (CD146, CD34, neural/glial antigen 2 [NG2], platelet-derived growth factor receptor-β [PDGFRβ], and α-smooth muscle actin [α-SMA]) in relation to endothelial markers (CD31, CD144, von Willebrand factor [vWF]). Pericytes and adventitial cells were isolated from the stromal vascular fraction (3.8% and 21.2%, respectively) using flow cytometry with a viability of 88%. The mean numbers of pericytes and adventitial cells isolated were 4.6 ± 2.2 × 104 and 16.2 ± 3.2 × 104 , respectively, equating to 7.9 ± 4.4 × 103 and 20.8 ± 4.3 × 103 cells per gram of harvested tissue. Fluorescence-activated cell sorting demonstrated that cultured PSCs were CD44+CD90+CD105+; polymerase chain reaction and immunocytochemistry demonstrated that pericytes retained their CD146+ phenotype and expressed the pericyte markers PDGFRβ and NG2. Differentiation was confirmed using histochemical stains and genetic expression. Using a pellet model, the IFP PSCs and the MSCs generated significantly more extracellular matrix than bone marrow MSCs (p < .001 and p = .011, respectively). The IFP PSCs generated significantly more extracellular matrix than IFP MSCs (p = .002). Micromass culture demonstrated that differentiated PSCs were upregulated compared with MSCs for COL2A1, ACAN, and SOX9 expression by factors of 4.8 ± 1.3, 4.3 ± 0.9, and 7.0 ± 1.7, respectively. The IFP was a significantly better source of chondrogenic stem cells compared with bone marrow. PSCs generated significantly more extracellular matrix than culture-derived MSCs. Stem Cells Translational Medicine 2017;6:77-87.

Kidney aging is accompanied by characteristic changes in the glomerulus, but little is known about the effect of aging on glomerular parietal epithelial cells (PECs), nor if the characteristic glomerular changes in humans and rats also occur in very old mice. Accordingly, a descriptive analysis was undertaken in 27-mo-old C57B6 mice, considered advanced age. PEC density was significantly lower in older mice compared with young mice (aged 3 mo), and the decrease was more pronounced in juxtamedullary glomeruli compared with outer cortical glomeruli. In addition to segmental and global glomerulosclerosis in older mice, staining for matrix proteins collagen type IV and heparan sulfate proteoglycan were markedly increased in Bowman's capsules of older mouse glomeruli, consistent with increased extracellular matrix production by PECs. De novo staining for CD44, a marker of activated and profibrotic PECs, was significantly increased in aged glomeruli. CD44 staining was more pronounced in the juxtamedullary region and colocalized with phosphorylated ERK. Additionally, a subset of aged PECs de novo expressed the epithelial-to-mesenchymal transition markers α-smooth muscle and vimentin, with no changes in epithelial-to-mesenchymal transition markers E-cadherin and β-catenin. The mural cell markers neural/glial antigen 2, PDGF receptor-β, and CD146 as well as Notch 3 were also substantially increased in aged PECs. These data show that mice can be used to better understand the aging kidney and that PECs undergo substantial changes, especially in juxtamedullary glomeruli, that may participate in the overall decline in glomerular structure and function with advancing age.

Human mesenchymal stem cells (hMSCs) are intrinsically heterogeneous and comprise subpopulations that differ in their proliferation, multi-potency, and functional properties, which are commonly demonstrated by culturing hMSCs at different plating densities. The objective of this study was to investigate the metabolic profiles of different subpopulations of hMSC by testing the hypothesis that the clonogenic hMSC subpopulation, which is selectively enriched in clonal density (CD) and low density (LD) culture (10 and 100 cells per square centimeter, respectively), possesses a metabolic phenotype that differs from that of hMSC in medium- or high-density (MD: 1,000 and HD: 3,000 cells per square centimeter, respectively). Cells at CD and LD conditions exhibited elevated expression of CD146 and colony forming unit-fibroblast compared with cells at MD- or HD. Global metabolic profiles revealed by gas chromatography-mass spectrometry of cell extracts showed clear distinction between LD and HD cultures, and density-dependent differences in coupling of glycolysis to the TCA cycle. Metabolic inhibitors revealed density-dependent differences in glycolysis versus oxidative phosphorylation (OXPHOS) for ATP generation, in glutamine metabolism, in the dependence on the pentose phosphate pathway for maintaining cellular redox state, and sensitivity to exogenous reactive oxygen species. We also show that active OXPHOS is not required for proliferation in LD culture but that OXPHOS activity increases senescence in HD culture. Together, the results revealed heterogeneity in hMSC culture exists at the level of primary metabolism. The unique metabolic characteristics of the clonogenic subpopulation suggest a novel approach for optimizing in vitro expansion of hMSCs.

α4-laminins, such as laminins 411 and 421, are mesenchymal laminins expressed by blood and lymphatic vessels and some tumor cells. Laminin-411 promotes migration of leukocytes and endothelial cells, but the effect of this laminin and laminin-421 on tumor cells is poorly understood. In the present study, we demonstrate that laminin-411 and, to a greater extent, laminin-421 significantly promote migration of tumor cells originated from melanomas, gliomas and different carcinomas via α6β1 integrin. In solid-phase binding assays, both laminins similarly bound α6β1 integrin but only laminin-421, among several laminin isoforms, readily bound MCAM (CD146), a cell-surface adhesion molecule strongly associated with tumor progression. Accordingly, a function-blocking mAb to MCAM inhibited tumor cell migration on laminin-421 but not on laminins 411 or 521. In tumor tissues, melanoma cells co-expressed MCAM, laminin α4, β1, β2 and γ1 chains, and integrin α6 and β1 chains. The present data highlight the novel role of α4-laminins in tumor cell migration and identify laminin-421 as a primary ligand for MCAM and a putative mediator of tumor invasion and metastasis.

Cartilage-derived mesenchymal stem cells (MSCs) have been isolated with different methods. In this study lateral and medial femoral condyles were respectively collected from patients with late-stage osteoarthritis during the total knee arthroplasty. After digestion of the cartilage tissues with type II collagenase and analysis by fluorescence-activated cell sorting (FACS) with CD146, a chondroprogenitor cell sub-population were isolated and purified. The expression of other MSC-associated markers in the CD146+ chondroprogenitors was analyzed by flow cytometry. Multi-lineage differentiation capacity of CD146+ chondroprogenitors was compared with that of unsorted chondrocytes and adipose-derived MSCs (ADMSCs). Higher percentage of CD146+ chondroprogenitors isolated from the medial femoral condyles was observed than that from the lateral. CD146+ chondroprogenitors expressed high levels of MSC-specific surface antigens, and showed higher chondrogenesis capacity than ADMSCs and unsorted chondrocytes in a 3D cell pellet culture model. Thus CD146 might be a new cell surface marker for cartilage progenitor cell population in the late-stage osteoarthritis.

Stem cells extracted from developing tissues possibly exhibit not only unique but also superior traits against their developed counterparts. Indeed, stem cells from the apical papilla (SCAP); a unique group of dental stem cells related to developing roots have been shown to be a promising tool for regenerative endodontic procedures and regeneration in general. Studies have characterized the phenotypic traits as well as other regenerative potentials of these cells. Specific sub-populations have been highlighted as well as their neurogenic and angiogenic properties. Nevertheless, in light of the previously discussed features and potential applications of SCAP, there is still much to understand and a lot of information to unravel. The current review will discuss the role of specific markers for detection of different functional populations of SCAP; including CD146 and STRO-1, as well as their true multilineage differentiation potential. In particular, the role of the secretome in association with paracrine signaling in inflammatory microenvironments is also tackled. Additionally, the role of SCAP both in vitro and in vivo during regenerative approaches and in response to different growth factors and biologic scaffolds is highlighted. Finally, this review will shed light on current knowledge regarding the clinical translational potential of SCAP and elucidate possible areas for future research applications.

BACKGROUND

Multipotent stem cells have been successfully isolated from various tissues and are currently utilized for tissue-engineering and cell-based therapies. Among the many sources, skin has recently emerged as an attractive source for multipotent cells because of its abundance. Recent literature showed that skin stromal cells (SSCs) possess mesoderm lineage differentiation potential; however, the endothelial differentiation and angiogenic potential of SSC remains elusive. In our study, SSCs were isolated from human neonatal foreskin (hNFSSCs) and adult dermal skin (hADSSCs) using explants cultures and were compared with bone marrow (hMSC-TERT) and adipose tissue-derived mesenchymal stem cells (hADMSCs) for their potential differentiation into osteoblasts, adipocytes, and endothelial cells.

RESULTS

Concordant with previous studies, both MSCs and SSCs showed similar morphology, surface protein expression, and were able to differentiate into osteoblasts and adipocytes. Using an endothelial induction culture system combined with an in vitro matrigel angiogenesis assay, hNFSSCs and hADSSCs exhibited the highest tube-forming capability, which was similar to those formed by human umbilical vein endothelial cells (HUVEC), with hNFSSCs forming the most tightly packed, longest, and largest diameter tubules among the three cell types. CD146 was highly expressed on hNFSSCs and HUVEC followed by hADSSCs, and hMSC-TERT, while its expression was almost absent on hADMSCs. Similarly, higher vascular density (based on the expression of CD31, CD34, vWF, CD146 and SMA) was observed in neonatal skin, followed by adult dermal skin and adipose tissue. Thus, our preliminary data indicated a plausible relationship between vascular densities, and the expression of CD146 on multipotent cells derived from those tissues.

CONCLUSIONS

Our data is the first to demonstrate that human dermal skin stromal cells can be differentiated into endothelial lineage. Hence, SSCs represents a novel source of stem/stromal cells for tissue regeneration and the vascularization of engineered tissues. Moreover, the CD146 investigations suggested that the microenvironmental niche might contribute to direct stromal cells multipotency toward certain lineages, which warrants further investigation.

OBJECTIVE

We attempted to evaluate endothelial dysfunction and the role of AECAs in systemic lupus (SL) with low disease activity. We quantified endothelial microparticles (EMps) and attempted to find the best flow cytometry method for that purpose.

METHODS

CD105, CD144 and CD146 were tested, individually or in combination, on EMp-enriched plasma. Twenty-three healthy blood donors and 27 SL patients were evaluated. SL patients with a SLEDAI <10 (median 2.6) were evaluated in our outpatient clinic. For each patient, EMps (CD105-CD146(+), CD45(-)) and AECAs were quantified and characterized.

RESULTS

The monochrome composite marker CD105-CD146 appeared to be the most efficient in detecting EMps. SL patients had more circulating EMps than healthy donors: respective median values were 2575 and 130 EMps/microl (P < 0.001). SL patients had more CD54(+) and CD54(-) EMps than healthy donors (496 vs 34 EMps CD54(+)/microl, P < 0.0001; 1875 vs 89 EMps CD54(-)/microl, P < 0.0001). The ratio CD54(+) EMps/total EMps was lower for lupus patients than for healthy individuals (20.3 vs 33.7%, P = 0.03). Twenty-four patients (89%) were positive for AECAs. EMp counts were not significantly higher for patients with AECAs.

CONCLUSIONS

Monochrome composite marker is efficient in detecting the whole population of EMps using flow cytometry. SL patients with low disease activity have a marked endothelial dysfunction. EMps released from the endothelium originate from activated and non-activated cells. AECAs do not seem to be the main cause of endothelial dysfunction in this population.

BACKGROUND

The aim of this study was to isolate and grow cells from sound human deciduous teeth pulp with different levels of resorption and evaluate stem cell parameters.

METHODS

Pulp tissue was removed from 30 different patients, aged from 6 to 12 years. From all the teeth, 21 were in advanced levels of resorption (group 1), and the remaining nine teeth did not show any visible resorption (group 2). Pulp tissue was removed and dissociated, and the suspension was seeded onto 12-well plates. The phenotype of the cells (n = 5) was analyzed on fifth and tenth passages by flow cytometry for clusters of differentiation (CD)29/PE, CD34/PE, CD44/FITC, CD45/FITC, CD90/FITC, CD117/PE, CD133/PE, CD146/FITC, CD184/PE, Stromal Cell Surface Marker 1 (STRO-1)/FITC and human leukocyte antigen major histocompatibility complex class II surface receptor (HLA-DR)/FITC, and by reverse transcription-polymerase chain reaction (RT-PCR) for octamer-binding transcription factor 4 (OCT-4). On the same passages, cells were differentiated into adipocytes, osteoblasts, and chondrocytes.

RESULTS

Cell isolation was successful in 25 samples, but only 17 of these reached 90% confluence. It was not possible to establish cell culture from group 2. Cells on both fifth and tenth passages were positive for CD29, CD44, and CD90 and also for the expression of OCT-4. Moderate labeling was observed for CD117 and CD133, whereas a low expression was detected for CD34, CD45, HLA-DR, CD184, CD146, and STRO-1. All cultures differentiated into three cell types.

CONCLUSIONS

The isolated pulp cells can be considered stem cells. The facility for obtaining cells seems to be related to the root resorption process, so, therefore, the cells from group 1 were able to proliferate in vitro, whereas group 2 cells were not.

Human mesenchymal stromal cells (hMSCs) have generated significant interest due to their potential use in clinical applications. hMSCs are present at low frequency in vivo, but after isolation can be expanded considerably, generating clinically useful numbers of cells. In this study, we demonstrate the use of a defined embryonic stem cell expansion medium, mTeSR (Stem Cell Technologies), for the expansion of bone-marrow-derived hMSCs. The hMSCs grow at comparable rates, demonstrate tri-lineage differentiation potential, and show similar surface marker profiles (CD29(+), CD44(+), CD49a(+), CD73(+), CD90(+), CD105(+), CD146(+), CD166(+), CD34(-), and CD45(-)) in both the fetal bovine serum (FBS)-supplemented medium and mTeSR. However, expression of early differentiation transcription factors runt-related transcription factor 2, sex-determining region Y box 9, and peroxisome proliferator-activated receptor gamma changed significantly. Both runt-related transcription factor 2 and sex-determining region Y box 9 were upregulated, whereas peroxisome proliferator-activated receptor gamma was downregulated in mTeSR compared with FBS. Although osteogenic and chondrogenic differentiation was comparable in cells grown in mTeSR compared to FBS, adipogenic differentiation was significantly decreased in mTeSR-expanded cells, both in terms of gene expression and absolute numbers of adipocytes. The removal of the FBS from the medium and the provision of a defined medium with disclosed composition make mTeSR a superior study platform for hMSC biology in a controlled environment. Further, this provides a key step toward generating a clinical-grade medium for expansion of hMSCs for clinical applications that rely on osteo- and chondroinduction of MSCs, such as bone repair and cartilage generation.

The field of regenerative medicine has increasingly recognized the importance to be inspired by developmental processes to identify signaling pathways crucial for 3D organogenesis and tissue regeneration. Here, we aimed at recapitulating the first events occurring during limb development (ie, cell condensation and expansion of an undifferentiated mesenchymal cell population) to prime 3D cultures of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) toward the chondrogenic route. Based on embryonic development studies, we hypothesized that Wnt3a and fibroblast growth factor 2 (FGF2) induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated β-catenin levels. Wnt3a was able to induce a five-fold increase in the number of proliferating hBM-MSC (6.4% vs. 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved transforming growth factor-β1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146⁺ subpopulation was found to be more responsive to Wnt3a. The presented data indicate a possible strategy to prime 3D cultures of hBM-MSC by invoking a "developmental engineering" approach. The study also identifies some opportunities and challenges to cross-fertilize skeletal development models and 3D hBM-MSC culture systems.

OBJECTIVE

An assay proposed to quantify endothelial progenitor cell (EPC) colonies in humans was investigated to determine the phenotype of recovered cells and their relevance to in vivo endothelial function.

RESULTS

Twelve sedentary subjects participating in a worksite wellness program underwent endothelial flow-mediated dilation (FMD) testing of the brachial artery and blood sampling for EPC colony assay. Microarray-based genotypic characterization of colonies showed surface markers consistent with T lymphocyte phenotype, but not with an EPC (CD34, CD133, VEGFR-2) or endothelial (CD146) phenotype. Gene expression patterns more closely matched T lymphocytes (r=0.87) than endothelial cells (r=0.66) in our microarray database. Flow cytometry of colonies confirmed large populations of CD3+CD45+ T cells (>75%) and few CD146+CD45- endothelial cells (<1%). Further, there was no correlation between colony number and the magnitude of FMD (r=-0.1512, P=0.6389). After exercise training, subjects improved FMD, from 6.7+/-2.0 to 8.7+/-1.9% (P=0.0043). Colonies also increased (P=0.0210), but without relation to FMD (r=0.1074, P=0.7396). T lymphocyte phenotype persisted after exercise (r=0.87).

CONCLUSIONS

Cells in a commonly used EPC colony assay have a gene expression and cell surface marker profile consistent with a predominance of T lymphocytes and have an unclear relevance to endothelial function, either before or after exercise training.

Recent studies have demonstrated that human dental pulp contains adult stem cells. A pulse of the thymidine analog BrdU given to young animals at the optimal time could clarify where slow-cycling long-term label-retaining cells (LRCs), putative adult stem cells, reside in the pulp tissue. This study focuses on the mapping of LRCs in growing teeth and their regenerative capacity after tooth injuries. Two to seven peritoneal injections of BrdU into pregnant Wistar rats revealed slow-cycling long-term dense LRCs in the mature tissues of born animals. Numerous dense LRCs were postnatally decreased in number and reached a plateau at 4 weeks after birth when they mainly resided in the center of the dental pulp, associating with blood vessels. Mature dental pulp cells were stained with Hoechst 33342 and sorted into (<0.76%) side population cells using FACS, which included dense LRCs. Some dense LRCs co-expressed mesenchymal stem cell markers such as STRO-1 or CD146. Tooth injuries caused degeneration of the odontoblast layer, and newly differentiated odontoblast-like cells contained LRCs. Thus, dense LRCs in mature pulp tissues were supposed to be dental pulp stem cells possessing regenerative capacity for forming newly differentiated odontoblast-like cells. The present study proposes the new hypothesis that both granular and dense LRCs are equipped in the dental pulp and that the dense LRCs with proliferative capacity play crucial roles in the pulpal healing process following exogenous stimuli in cooperation with the granular LRCs.

Human periodontal ligament stem cells (PDLSCs) are a unique population of mesenchymal stem cells (MSCs) which demonstrate the capacity to generate cementum- and periodontal ligament-like structures in vivo. As such, PDLSCs represent a promising cell-based therapy in reconstructive dentistry for the treatment of periodontal disease. The present chapter describes two methods for isolating PDLSCs from human PDL tissue including traditional plastic adherence and immunomagnetic selection based on the expression of MSC-associated surface markers STRO-1 antigen, CD146 (MUC-18), CD29 (integrin beta-1), CD44, and CD106 (VCAM-1). Although no single antibody demonstrates specificity for MSCs, isolation based on the expression of individual markers results in homogeneous preparations of PDLSCs. Methods to further characterize the immunophenotype and multipotent capacity of PDLSCs to differentiate into adipocytes, osteoblast- and cementoblast-like cells in vitro, and cementum- and periodontal ligament-like tissues in vivo are also described.