Hematopoietic Cell Markers CD34 Research Articles

Mesenchymal Stem Cell Identification After Delayed Cord Clamping.

We sought to determine the feasibility of identifying and quantifying mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) after delayed cord clamping in preterm and term births. We obtained 3mL of UCB at various gestational ages after delayed cord clamping. UCB separated by density gradient centrifugation within 4h of delivery was passed through magnetic bead micro-columns to exclude the CD34 + cell population. The samples were incubated with fluorescent-tagged mesenchymal cell marker antibodies CD 29, CD44, CD73, CD105, and hematopoietic cell marker CD45. The cell populations were analyzed by flow cytometry. Viable cells were assessed with 7-aminoactinomycin-D. The results were expressed in median (minimum to maximum) MSCs and compared between preterm and term samples. A total of 12 UCB samples (32-40weeks) were obtained, 10 of which demonstrated MSCs, accounting for 0.0174% (0-14.7%) of the viable UCB mononuclear cells. MSCs comprised 0.148% (0.0006-1.59%) and 0.116% (0-14.7%) of the viable UCB mononuclear cells in the term (n = 5), 38.4 ± 1.3weeks, and preterm (n = 7) samples, 34.6 ± 1.1, respectively, p = 0.17. There was an overall median of 96 (0-39,574) MSCs. There was no difference in the median numbers of MSCs identified between term and preterm UCB samples, 3384 (23-6042) and 36 (0-39,574), respectively, p = 0.12. Mesenchymal stem cells were identified and quantified in 5 of 7 preterm and all 5 term UCB 3-mL samples obtained after delayed cord clamping.

A flow cytometric method for measuring and isolating mammary epithelial cells from bovine milk

Sampling frequent time points of mammary signaling pathways is not possible with tissue biopsies. We have validated a flow cytometry and cell sorting procedure for isolating live bovine mammary epithelial cells from somatic cell populations in milk using butyrophilin 1A1 as a marker for mammary epithelial cells and CD45 as a marker for hematopoietic cells. Hoechst 33342 staining and propidium iodide exclusion were used to select for nucleated live cells. Positive selection of butyrophilin (BTN)-expressing cells was performed by fluorescence-activated cell sorting. Quantitative real-time PCR performed on mRNA isolated from these cells showed a 226-fold increase in κ-casein (CSN3) mRNA expression in BTN single-positive cells compared with unsorted cells, whereas CD45 single-positive cells showed a significant decrease. A negative selection strategy for cells not expressing the hematopoietic cell marker CD45 also resulted in a cell population with a 196-fold increase in CSN3 mRNA expression compared with unsorted cells. We found no enrichment of CSN3 mRNA expression after sorting cells using cytokeratin antibodies. The noninvasive assays described here allow for daily or more frequent sampling time points for measurement of mammary epithelial cells during the course of lactation.

Fibrocyte accumulation in the lungs of cystic fibrosis patients

BackgroundCystic fibrosis (CF) patients develop severe lung disease including chronic airway infections, neutrophilic inflammation, and progressive fibrotic remodeling in airways. However, cellular and molecular processes that regulate excessive collagen deposition in airways in these patients remain unclear. Fibrocytes are bone marrow (BM)-derived mesenchymal cells that express the hematopoietic cell marker CD45, and mesenchymal cell markers and implicated in collagen deposition in several fibrotic diseases. It is unknown whether fibrocytes accumulate in the lungs of CF patients, so the current study evaluates the presence of fibrocytes in the fibrotic lesions of airways in explanted CF lungs compared to non-CF unused donor lungs (control). MethodsWe used immunofluorescence staining to determine if fibrocytes accumulate in explanted CF lungs compared to healthy donor lungs. Simultaneously, we evaluated cells collected by bronchoalveolar lavage (BAL) in CF patients using multi-color flow cytometry. Finally, we analyzed transcripts differentially expressed in fibrocytes isolated from the explanted CF lungs compared to control to assess fibrocyte-specific pro-fibrotic gene networks. ResultsOur findings demonstrate fibrocyte accumulation in CF lungs compared to non-CF lungs. Additionally, fibrocytes were detected in the BAL of all CF children. Transcriptomic analysis of fibrocytes identified dysregulated genes associated with fibrotic remodeling in CF lungs. ConclusionsWith significantly increased fibrocytes that show increased expression of pro-fibrotic gene transcripts compared to control, our findings suggest an intervention for fibrotic remodeling as a potential therapeutic target in CF.

Post-calving umbilical cord tissue offcut: A potential source for the isolation of bovine mesenchymal stem cells.

Background and Aim:Veterinary health care is an emergent area in animal sciences and innovative therapeutic approaches happen to be imperative in the present days. In view of the importance of cattle health and production, it is necessary to take up contemporary approach of stem cell therapy in this sector also. This study aimed to standardize an explant culture method of bovine umbilical tissue offcut to isolate mesenchymal stem cells (MSCs) because considerable efforts are required for ensuring easy accessibility and availability of MSCs in bulk quantity, as well as in establishing and characterizing the cell lines.Materials and Methods:The umbilical cord (UC) tissue matrix offcut was collected after calving. A simplified in vitro cell isolation technique was followed to collect the emerged out cells from the explants of UC. Further, we expanded these isolated cells in vitro, observed its growth kinetics, and characterized to confirm as per the criterion of bovine MSCs.Results:A considerable exponential growth rate of the UC-derived cells was noticed. In addition to their confirmation as MSCs, the cells also exhibited plastic adherent property and maintained the spindle-shaped morphology throughout the in vitro culture. The cultured cells were found positive MSC-specific surface markers CD105, CD90, and CD73 and were negative for hematopoietic cell marker CD45. Cytochemical studies revealed the ability of the cells to differentiate into osteogenic, chondrogenic, and adipogenic lineages.Conclusion:This simplified method of isolation and culture of bovine multipotent MSCs from the UC offcut collected after calving could be extrapolated for the greater availability of the cells for prospective therapeutic applications.

Cryopreservation and multipotential characteristics evaluation of a novel type of mesenchymal stem cells derived from Small Tailed Han Sheep fetal lung tissue

Lung mesenchymal stem cells (L-MSCs) characterized by plasticity, reduced relative immune privilege and high anti-fibrosis characteristics play the crucial role in lung tissue regenerative processes. However, up to date, the multi-differentiation potentials and application values of L-MSCs are still uncertain. In the current study, the Small Tailed Han Sheep embryo L-MSCs line from 12 samples, stocking 124 cryogenically-preserved vials, was successfully established by using primary culture and cell cryopreservation techniques. Isolated L-MSCs were morphologically consistent with fibroblasts, could be passaged for at least 18 passages and more than 91.8% of cells were diploid (2n = 54) analyze by G-banding. The majority of cells were in the G0/G1 phase (70.5–91.2%), and the growth curves were all typically sigmoidal. Moreover, L-MSCs were found to express pluripotent genes Oct4, Nanog and MSCs-associated genes β-integrin, CD29, CD44, CD71, CD73 and CD90, while the expressions of hematopoietic cell markers CD34 and CD45 were negative. In addtion, the L-MSCs could be differentiated into cells of three layers with induction medium in vitro, which confirmed their multilineage differentiation potential. The secretion of urea and ALB showed the differentiated hepatocytes still possessed the detoxification function. These results indicated that the isolated L-MSCs displayed typical characteristics of mesenchymal stem cells and that the culture conditions were suitable for their maintenance of stemness and their proliferation in vitro.

Influence of different types of pulp treatment during isolation in the obtention of human dental pulp stem cells.

BackgroundDifferent methods have been used in order to isolate dental pulp stem cells. The aim of this study was to study the effect of different types of pulp treatment during isolation, under 3% O2 conditions, in the time needed and the efficacy for obtaining dental pulp stem cells.Material and MethodsOne hundred and twenty dental pulps were used to isolate dental pulp stem cells treating the pulp tissue during isolation using 9 different methods, using digestive, disgregation, or mechanical agents, or combining them. The cells were positive for CD133, Oct4, Nestin, Stro-1, CD34 markers, and negative for the hematopoietic cell marker CD-45, thus confirming the presence of mesenchymal stem cells. The efficacy of dental pulp stem cells obtention and the minimum time needed to obtain such cells comparing the 9 different methods was analyzed.ResultsDental pulp stem cells were obtained from 97 of the 120 pulps used in the study, i.e. 80.8% of the cases. They were obtained with all the methods used except with mechanical fragmentation of the pulp, where no enzymatic digestion was performed. The minimum time needed to isolate dental pulp stem cells was 8 hours, digesting with 2mg/ml EDTA for 10 minutes, 4mg/ml of type I collagenase, 4mg/ml of type II dispase for 40 minutes, 13ng/ml of thermolysine for 40 minutes and sonicating the culture for one minute.ConclusionsDental pulp stem cells were obtained in 97 cases from a series of 120 pulps. The time for obtaining dental pulp stem cells was reduced maximally, without compromising the obtention of the cells, by combining digestive, disgregation, and mechanical agents. Key words:Dental pulp stem cells, mesenchymal stem cells, isolation method.

Inhibition of bone marrow mesenchymal stem cell autologous transplantation on inflammation following rabbit corneal alkali burn

Background Ocular alkali burns leads to corneal ulcer and angiogenesis and even corneal opacity. There is still no ideal treatment method. Studies showed that mesenchymal stem cells (MSCs) can repair corneal wound in vivo, but the specific mechanism is still not clear. Objective This study aimed to observe the histopathological change after the early transplatation of bone marrow MSCs (BMSCs) for corneal alkali burn model in rabbits and explore the anti-inflammatory effects of MSCs after corneal alkali burn. Methods Bone marrow of 4 ml was collected from 2-3 month-old Japanese rabbit. BMSCs were isolated and cultured from the bone marrow of rabbits, and the third generation of cells were used in this study. Cultured cells were identified by morphology and the expressions of surface markers. Corneal alkali burn models were extablished in the right eyes of 24 rabbits by attaching the filter paper with 0. 1% NaOH at the central cornea for 30 seconds, and then the models were randomized into 2 groups. BMSCs suspension of 300 μl (concentration 5×106/μl) was subconjunctivally injected 1 hour after modeling in the BMSCs group, and equal volume of PBS was used in the same way in the PBS group. Corneal opacification was scored under the slim lamp microscope in 3, 14 and 28 days after injection. The polymorphonuclear neutrophils (PMNs) were counted by histopathological examination, and the expression of matrix metalloproteinase-2 (MMP-2) in the corneal tissue was evaluated by immunochemistry in various time points. The use and care of the rabbits followed the statement of ARVO. Results The rabbit BMSCs were plastic-adherent cells that exhibited a fibroblast-like shape. Cultrued cells highly expressed surface adhesion molecular markers CD29 and CD90 (99. 18% and 97. 94%) and lowly expressed hematopoietic cell markers CD34 and CD31 (0. 74% and 0. 15%). Opacification of cornea, defect of corneal epithelium, stromal edema and neovascularization appeared after modeling. In 14 days and 28 days after modeling, the opacification scores in the BMSCs group were 2. 37±0. 52 and 2. 25±0. 50, which were significantly lower than 3. 00±0. 53 and 3. 25±0. 50 in the PBS group (t=2. 376, 2. 828, both at P<0. 05). After subconjunctival injection, the number of PMNs was (34. 17±1. 85) /12 fields and (25. 64±3. 86)/12 fields in the BMSCs group, showing significant decrease in comparison with (42. 70±1. 54) /12 fields and (32. 67±1. 42)/12 fields in the PBS group (t=10. 021, 4. 832, both at P=0. 000). The expression levels of MMP-2 (A value) in cornea were 0. 388±0. 016 and 0. 384±0. 006 in the BMSCs group, with considerable decreases in comparison with 0. 438±0. 006 and 0. 412±0. 005 in the PBS group (t=10. 205, 13. 514, both at P=0. 000). Conclusions Early transplantation of BMSCs can arrest the occurrance of corneal ulcer by suppressing the infiltration of PMNs, alleviateing the inflammation reaction, downregulating the expression of MMP-2 in cornea and inhibiting the degradation of stromal collagen fibers. Key words: Bone marrow mesenchymal stem cell transplantation/methods; Biological markers; Burns, chemical/therapy; Corneal diseases/therapy; Neovascularization, pathologic/chemically induced; Inflammation/prevention & control; Matrix metalloproteinase; Disease models, animal

The herbal compound "diwu yanggan" modulates liver regeneration by affecting the hepatic stem cell microenvironment in 2-acetylaminofluorene/partial hepatectomy rats.

Ethnopharmacological Relevance. “Diwu Yanggan” (DWYG) has been reported to regulate liver regeneration, modulate the immune response, ameliorate liver injury, kill virus, ameliorate liver fibrosis, and suppress hepatic cancer. However, its mechanisms are still unknown. Objectives. To investigate the effects of DWYG on oval cell proliferation in 2-AAF/PH rats and determine its mechanism. Methods. Wistar rats were randomly distributed into normal group, sham group, vehicle group, and DWYG group. Hepatic pathological changes were examined by H&E staining. The oval cell markers CD34, AFP, CK-19 and hematopoietic cell markers CD45, Thy1.1, and hepatocyte marker ALB were examined with immunohistochemistry. The percentage of CD34/CD45 double-positive cells in bone marrow was detected by flow cytometry. Cytokine levels were measured with the Bio-plex suspension array system. Results. DWYG significantly increased the survival rates of 2-AAF/PH rats and promoted liver regeneration. Furthermore, DWYG increased the ratio of CD34/CD45 double-positive cells on days 10 and 14. In addition, DWYG gradually restored IL-1, GRO/KC, and VEGF levels to those of the normal group. Conclusions. DWYG increases 2-AAF/PH rat survival rates, suppresses hepatic precarcinoma changes, and restores hepatic tissue structure and function. DWYG may act by modulating the hepatic microenvironment to support liver regeneration.

Bone Marrow Mesenchymal Stem and Progenitor Cells Are Altered in Sipa1 Deficient Mice before Initiation of Myeloproliferative Neoplasm

Hematopoiesis is tightly regulated by bone marrow (BM) stromal cells through the production of soluble factors, matrix proteins, and intracellular signals initiated by cell-cell adhesive interactions. Despite the involvement of mesenchymal stem and progenitor cells in the maintenance of normal hematopoiesis and the development of hematological disorders have been reported (Mendez-Ferrer et al., 2010, Nature)(Raaijmakers et al., 2010, Nature), little is known how the different BM stromal cell fractions contribute to the processes.Signal-induced proliferation-associated gene 1 (Sipa1), a principal Rap1 GTPase activating protein, is expressed in hematopoietic cells (Ishida et al., 2003, Cancer Cell). Deletion of Sipa1 results in age-dependent development of myeloproliferative neoplasm (MPN) in mice, resembling human chronic myelogenous leukemia (Ishida et al., 2003, Cancer Cell). However, it is not known whether the deletion of Sipa1 affects BM cellular niche components, which could lead to the development of the MPN. Knowledge of this is important for better understanding the role of BM stromal cells in the initiation and progression of MPN.In the present study, we first analyzed Sipa1 expression by multicolor fluorescent activated cell sorting (FACS) using hematopoietic cell marker CD45 and TER119, mesenchymal stem cell (MSC) associated marker CD51 and SCA1, and endothelial cell marker CD31. We found that Sipa1 gene was also expressed in different BM stromal cell subsets including MSCs (CD45-TER119-CD31-CD44-CD51+SCA1+), more differentiated mesenchymal progenitor cells (CD45-TER119-CD31-CD44-CD51+SCA1-), and endothelial cells (CD45-TER119-CD31+). Among all the cell types, Sipa1 expression seemed to be higher in MSCs and endothelial cells than other cell types. We next studied whether the gene deficiency led to the alteration of BM stromal cell composition and function by phenotypically, functionally, and molecularly analyzing BM stromal cell subsets in young (<3 months) Sipa1-/- mice where hematopoiesis remained normal. Phenotypic multicolor FACS analysis showed significant increase in percentages and numbers of BM stromal cell (CD45-TER119-) subsets including endothelial cells, MSCs, and the mesenchymal progenitor cells in Sipa1-/- mice compared to age and sex-matched Sipa1+/+ mice (P<0.05). The alteration in the frequency of BM MSCs in the Sipa1-/- mice was further confirmed by colony-forming unit-fibroblast assay (CFU-F) on unfractionated BM mononuclear cells (P=0.0302) and FACS-sorted MSCs (P=0.0066). It is important to note that most (75% on average) of CFU-Fs generated from FACS-sorted Sipa1-/- stromal cells contained simultaneously differentiated adipocytes whereas around 57% of CFU-Fs from Sipa1+/+ stromal cells did (P=0.0184). In keeping with this, in vitro differentiation assays revealed greater adipocyte differentiation potential of Sipa1-/- MSCs. Similarly, an increase of adipocytes associated gene FABP4 expression was determined by Q-PCR. Our preliminary Q-PCR data indicated differential expression of Kit Ligand and Angipoietinlike 1 in the Sipa1-/- MSCs and the CD51+SCA1- cells compared to that in the Sipa1+/+ cells. Global gene expression analysis by RNA sequencing and more multilineage differentiation assays on FACS-sorted different stromal cell subsets of Sipa1-/- and Sipa1+/+ BM are currently ongoing.Taken together, our data suggested the BM stromal cell composition and function in the Sipa1-/- mice have already been altered before the initiation of MPN. Transplantations of normal hematopoietic stem cells into Sipa1 deficient BM have been initiated for examining if the Sipa1-/- BM niches play an instructive role in the initiation of MPN. DisclosuresNo relevant conflicts of interest to declare.

Segregation of late outgrowth endothelial cells into functional endothelial CD34- and progenitor-like CD34+ cell populations.

Late outgrowth endothelial cells (OECs) that originate from peripheral blood mononuclear cells ex vivo have phenotypic and functional properties of mature endothelial cells. Given the potential therapeutic applications of OECs, understanding their biology is crucial. We have identified two distinct OEC populations based on differential expression of the cell surface marker CD34. OEC colonies lacked CD34 expression (CD34-), expressed CD34 in the majority of cells (CD34+), or showed a mixed expression pattern within a colony (CD34+/-). CD34+ and CD34- OECs were negative for hematopoietic cell marker CD45 and expressed the endothelial cell surface markers CD31, CD146, CD105, and VEGFR-2. Functionally CD34- and CD34+ OECs exhibited strikingly distinct behaviors. CD34- OECs, unlike CD34+ OECs, were capable of sprouting, formed tubes, and responded to angiogenic growth factors in vitro. In vivo, CD34- OECs formed endothelial tubes, while CD34+ OECs, despite being unable to form tubes, promoted infiltration of murine vasculature. Global gene expression profiling in CD34- and CD34+ OECs identified functional importance of the MMP-1/PAR-1 pathway in CD34- OECs. MMP-1 stimulated the expression of VEGFR-2, neuropilin-1, neuropilin-2, and CXCR4 and activated ERK1/2, whereas down-regulation of PAR-1 in CD34- OECs resulted in impaired angiogenic responses in vitro and reduced VEGFR-2 levels. In contrast, the CD34+ OEC colonies expressed high levels of the progenitor cell marker ALDH, which was absent in CD34- OECs. In summary, we show that OECs can be classified into functionally mature endothelial cells (CD34- OECs) that depend on the MMP-1/PAR-1 pathway and progenitor-like angiogenesis-promoting cells (CD34+ OECs).

Human Hepatic Progenitor Cells Express Hematopoietic Cell Markers CD45 and CD109

Objective: To clarify the precise characteristics of human hepatic progenitor cells (HPCs) for future cytotherapy in liver diseases.Methods: Hepatic progenitor-like cells were isolated and cultured from the livers of patients who had undergone partial hepatectomy for various pathologies but displayed no sign of hepatic dysfunction. These cells were characterized by transcriptomic profiling, quantitative real-time PCR and immunocyto/histochemistry.Results:Cultured HPCs contained polygonal, high nucleus/cytoplasm ratio and exhibited a global gene expression profile similar (67.8%) to that of primary hepatocytes. Among the genes with more than 20-fold higher expression in HPCs were a progenitor marker (CD90), a pentraxin-related gene (PTX3), collagen proteins (COL5A2, COL1A1 and COL4A2), cytokines (EGF and PDGFD), metabolic enzymes (CYBRD1, BCAT1, TIMP2 and PAM), a secreted protein (SPARC) and an endothelial protein C receptor (PROCR). Moreover, eight markers (ALB, AFP, CK8, CK18, CK19, CD90, CD117 and Oval-6) previously described as HPC markers were validated by qRT-PCR and/or immunocyto/histochemistry. Interestingly, human HPCs were also positive for the hematopoietic cell markers CD45 and CD109. Finally, we characterized the localization of HPCs in the canals of Hering and periportal areas with six previously described markers (Oval-6, CK8, CK18, CK19, CD90 and CD117) and two potential markers (CD45 and CD109).Conclusion: The human HPCs are highly similar to primary hepatocytes in their transcriptional profiles. The CD45 and CD109 markers could potentially be utilized to identify and isolate HPCs for further cytotherapy of liver diseases.

Hypoxia, Leptin, and Vascular Endothelial Growth Factor Stimulate Vascular Endothelial Cell Differentiation of Human Adipose Tissue-Derived Stem Cells

The plasticity of human adipose tissue-derived stem cells (hASCs) is promising, but differentiation in vitro toward endothelial cells is poorly understood. Flow cytometry demonstrated that hASCs isolated from excised fat tissue were positive for CD29, CD44, CD70, CD90, CD105, and CD166 and negative for the endothelial marker CD31, and the hematopoietic cell markers CD34 and CD133. hASCs differentiated into adipocytes after cultivation in adipogenic medium. Exposure of hASCs for 10 days under hypoxia (3% oxygen) in combination with leptin increased the percentage of CD31(+) endothelial cells as well as CD31, VE-Cadherin, Flk-1, Tie2, von Willebrand factor, and endothelial cell nitric oxide synthase mRNA expression. This was enhanced on co-incubation of vascular endothelial growth factor (VEGF) and leptin, whereas VEGF alone was not sufficient. Moreover, hASCs cultured on a matrigel surface under hypoxia/VEGF/leptin, showed a stable branching network. Hypoxic conditions significantly decreased apoptosis as evaluated by cleaved caspase-3, and increased prolyl hydroxylase domain 3 mRNA expression. Hypoxia increased expression of VEGF as well as leptin transcripts, which were significantly inhibited on co-incubation with either VEGF or leptin or a combination of both. Furthermore, leptin treatment of hypoxic cells increased the expression of the long/signaling form of the leptin receptor (ObRL), which was augmented on co-incubation with VEGF. The observed endothelial differentiation was dependent on the Akt pathway, as co-administration with Akt inhibitor abolished the observed effects. In conclusion, our data demonstrate that hASCs can be efficiently differentiated to endothelial cells by mimicking the hypoxic and pro-angiogenic microenvironment of adipose tissue.

Characterization of Anoikis-Resistant Cells in Mouse Colonic Epithelium

Anoikis is a form of apoptosis triggered by inadequate or inappropriate cell matrix contacts. Anoikis resistance has been utilized to isolate adult stem or progenitor cell populations from various tissues. The aim of this study was to characterize the stem or progenitor cell markers expressed in anoikis-resistant cells isolated from mouse colonic epithelium. Mouse colonic epithelial cells were isolated by Ca(2+)-depletion, and anoikis-resistant cells were obtained by culturing the cells in ultra-low attachment dishes. Flow cytometry analysis showed that anoikis-resistant cells are positive for the epithelial cell marker CD326, but negative for the hematopoietic cell marker CD45, eliminating the possibility of hematopoietic cell contamination. The majority of anoikis-resistant cells were also positive for the stem or progenitor cell markers CD133 and DCLK1 by immunofluorescent analysis. Reverse transcriptase-polymerase chain reaction analysis revealed that anoikis-resistant cells express +4 position cell marker Hopx, but did not express the other reported stem or progenitor cell markers, Lgr5, Musashi1, Bmi1, mTert and Olfm4. CD133 and DCLK1 double positive cells were observed both apical and basal crypts in mouse proximal colonic tissues. Together, anoikis-resistant cells in mouse colon epithelium were shown to be positive for CD133, DCLK1, Hopx and CD326, but negative for CD45, Lgr5, Musashi1, Bmi1, mTert and Olfm4. This study has shown a possible approach to isolating stem cells from the intestinal epithelium.

Expression of early hematopoietic markers in cord and mobilized blood

G-CSF mobilized peripheral blood and cord blood are major sources of hematopoietic progenitor cells. These cells are characterized by the expression of "early" antigens. We have evaluated the coexpression of hematopoietic cell markers CD34, CD133, CD90, CDCP1, CD117 and activation antigen CD38 using multicolor flow cytometry. We show that (1) cells being positive for every single antigen form a separate population. (2) Percentage of cells expressing each "early" antigen are twice more in the cord blood than in the mobilized blood. The content of cells with complex progenitor phenotype (CD34+/CD38-/CD117, CD133+/CD34+/CD38-, CDCP1+/CD34+/CD38- etc.) is equal in mobilized and cord blood. (3) There are strong positive correlations between the expression of CD34, CD133, CD117 and CDCP1 in both groups. Positive correlation exists for CD90 with CD34, CD133, CDCP1 and CD117 only in cord blood and is not significant in mobilized blood. The analyses of early antigens coexpression with activation marker CD38 revealed that hypothesis on sequential activation and loss of expression of the aforementioned antigens is not confirmed. We assume that there is global regulation of the expression of CD34, CD133, CDCP1 and CD117. Yet expression of CD38 could be reversibly abolished during maturation of the hemapoetic cells and CD117 could be expressed not only on myeloid cells.

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 cm(3)). Growth kinetics revealed a mean cell doubling time of 49.9 h and a high number of 2.54 x 10(7) 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 alpha-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.

VEGF Induces Differentiation of Functional Endothelium From Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) offer a sustainable source of endothelial cells for therapeutic vascularization and tissue engineering, but current techniques for generating these cells remain inefficient. We endeavored to induce and isolate functional endothelial cells from differentiating hESCs. To enhance endothelial cell differentiation above a baseline of approximately 2% in embryoid body (EB) spontaneous differentiation, 3 alternate culture conditions were compared. Vascular endothelial growth factor (VEGF) treatment of EBs showed the best induction, with markedly increased expression of endothelial cell proteins CD31, VE-Cadherin, and von Willebrand Factor, but not the hematopoietic cell marker CD45. CD31 expression peaked around days 10 to 14. Continuous VEGF treatment resulted in a 4- to 5-fold enrichment of CD31(+) cells but did not increase endothelial proliferation rates, suggesting a primary effect on differentiation. CD31(+) cells purified from differentiating EBs upregulated ICAM-1 and VCAM-1 in response to TNFalpha, confirming their ability to function as endothelial cells. These cells also expressed multiple endothelial genes and formed lumenized vessels when seeded onto porous poly(2-hydroxyethyl methacrylate) scaffolds and implanted in vivo subcutaneously in athymic rats. Collagen gel constructs containing hESC-derived endothelial cells and implanted into infarcted nude rat hearts formed robust networks of patent vessels filled with host blood cells. VEGF induces functional endothelial cells from hESCs independent of endothelial cell proliferation. This enrichment method increases endothelial cell yield, enabling applications for revascularization as well as basic studies of human endothelial biology. We demonstrate the ability of hESC-derived endothelial cells to facilitate vascularization of tissue-engineered implants.

Hematopoietic cell activation in the subventricular zone after Theiler's virus infection

BackgroundThe periventricular subventricular zone (SVZ) contains stem cells and is an area of active neurogenesis and migration. Since inflammation can reduce neurogenesis, we tested whether Theiler's murine encephalomyelitis virus (TMEV) induces inflammation and reduces neurogenesis in the SVZ.MethodsWe performed immmunohistochemistry for the hematopoietic cell marker CD45 throughout the central nervous system and then examined neuroblasts in the SVZ.ResultsCD45+ activation (inflammation) occurred early in the forebrain and preceded cerebellar and spinal cord inflammation. Inflammation in the brain was regionally stochastic except for the SVZ and surrounding periventricular regions where it was remarkably pronounced and consistent. In preclinical mice, SVZ neuroblasts emigrated into inflamed periventricular regions. The number of proliferating phoshpohistone3+ cells and Doublecortin+ (Dcx) SVZ neuroblasts was overall unaffected during the periods of greatest inflammation. However the number of Dcx+ and polysialylated neural cell adhesion molecule (PSA-NCAM+) SVZ neuroblasts decreased only after periventricular inflammation abated.ConclusionOur results suggest that after TMEV infection, the SVZ may mount an attempt at neuronal repair via emigration, a process dampened by decreases in neuroblast numbers.

Expression of hematopoietic cell markers by retinal pigment epithelial cells

PURPOSE. We investigated the expression of various isoforms of the hematopoietic cell marker CD45 on retinal pigment epithelial cells in relation to their expression of CD68 and the cytokine-reactive intercellular adhesion molecule-1 (ICAM-1). We also determined the effect of the pro- inflammatory cytokines IL-1ß, TNFa and IFN? on the expression of these molecules by RPE cells in culture. METHODS. Monolayers of RPE cells between 3rd and 7th passages were cultured in the presence or absence of cytokines, followed by immunohistochemical staining for CD45 (170–220 kD), CD45RA (205 and 220 kD), CD45RO (180 kD), CD68 and ICAM-1, using the alkaline phosphatase-anti-alkaline phosphatase (APAAP) technique. Total (membrane and cytoplasmic) expression of each of the three CD45 isoforms was determined by enzyme-linked immunoassays (ELISA). RESULTS. The majority of RPE cells expressed all isoforms of CD45 on their membranes and the pattern of expression of these molecules was not modified by culture. The greatest intensity of membrane staining was consistently observed with antibodies to CD45RA (205 + 220 kD), while CD45 (170–220 kD) showed to be the predominant isoform within the whole cell, as judged by ELISA assays. Unlike the membrane expression of CD45, only 20% of RPE cells stained for the macrophage surface molecule CD68 following 4 h of culture, but progressive increase in the proportion of CD68 positive cells was observed by extending the culture to 24 and 48 h. Neither the expression of CD68 nor the various isoforms of CD45 were modified by incubation with pro-inflammatory cytokines. Staining for ICAM-1 was observed in 21–25% of RPE cells throughout the 48 h culture. However, incubation with 50 pg/ ml of IL-1ß, TNFa and IFN? caused a marked increase in the RPE cell expression of ICAM-1 following 4, 24 and 48 h culture. CONCLUSIONS. The observations suggest that hematopoietic cell markers are constitutively expressed on RPE cells and that functions governed by these molecules are not influenced by pro-inflammatory signals. Expression of hematopoietic molecules by RPE cells may influence the macrophage-like properties of these cells and may also aid in the identification of RPE cells during pathological processes, particularly in the proliferative retinopathies, where these cells undergo phenotypic and functional changes.

Day 11 mouse fetal thymus: phenotype and search for the point of commitment

This phenotype study of uncommitted hematopoietic cells (UHC) and early T cell precursors (ETCP) in the thymus provides an understanding of the commitment process from UHC to ETCP. The study of genes that are involved in this process depends on the proper identification of these early cells. Nonetheless, most current phenotype studies of these cells are based on the observations from the late stages of fetal or adult thymocytes. Though conclusions drawn from these studies are insightful, cell maturation occurs so fast during thymus development that some important phenotypic nuances go unnoticed if one only looks at late-stage cells. Furthermore, even though early-stage thymocytes are phenotypically similar to those of late stage, they may have different properties. In order to study those thymus populations at the very beginning of their differentiation and commitment, 11-day mouse fetal thymuses were tested. This shows, using day-12 CFU-S as a measure, that at this stage of development there are UHC present. Thymocytes at this very initial stage of development are isolated for the first time, and their phenotypes as well as their differentiation potentials are analyzed. The result shows that UHC are detected only in the Mac-1 − C-kit + subset, which comprises 2% of the total thymus population. Surprisingly, 93% of the C-kit + population is Mac-1 +, which generates T cells with a frequency of 1/72. This indicates that the Mac-1 molecule is a differentiation marker rather than a lineage-specific marker. In addition, C-kit + thymocytes at this stage do not express any T cell markers, such as Thy1.2, IL-2Rα, CD2 and Mel-14. These thymocytes are very akin to UHC, since they express Sca-1, Wga, and CD34, and are likewise similar to ETCP, as they express Pgp high, Mhc-I high, Hsa low, and FcR low. Moreover, they express a high level of adhesion molecules such as Lfa-1, Icam-1 and Lpam-1. As expected, these C-kit + all contain the hematopoietic cell marker CD45. Low expression of CD4 (the typical marker associated to the earliest T cell precursor in adult thymuses) is also found in 10% of the C-kit + population. While the C-kit + population at this stage is more homogeneous than at any other stage of fetal thymus development, there are still markers (B220, CD5, Tsa, Mac-1, CD4, and Sca-1) that can split this population into other subsets. However, the majority of these markers are present in the Mac-1 + C-kit + population, indicating that the C-kit + population is essentially made of two populations (Mac-1 + and Mac-1 − subsets). Interestingly, two major single-positive populations appear to emerge from this C-kit + population one day later, namely Thy1.2 + IL-2Rα − and Thy1.2 − IL-2Rα +. These two major single-positive populations seem to be derived directly from the Mac-1 + rather than from the Mac-1 − subset of the C-kit + population. Thus, these data suggest that important phenotypes are present during the early differentiation process. These phenotypes have never been shown before. Hopefully, this study will open up a new avenue for the study of very early stage T cell sublineages and their relationship to uncommitted thymic hematopoietic cells.