The dental pulp represents an easily accessible source of adult dental pulp stem cells (DPSCs). The preferred approach to DPSC isolation is enzymatic digestion. However, the duration of the enzymatic activity is crucial. The purpose of this study was to isolate the DPSC populations using this method, characterize their biological properties and proliferation capacity, and to determine their ability to differentiate into mature cells. Before enzymatic digestion using 0.05% trypsin, we used the homogenization method in order to obtain a fine homogenate from the solid pulp tissue. The stem cells were cultivated in modified cultivation medium for mesenchymal adult progenitor cells containing 2% foetal bovine serum, growth factors and insulin-transferrin-selenium supplement. We were successfully able to isolate 10 populations of DPSCs. The vitality of DPSCs did not drop below 90 %. However, the DPSCs showed a significant decrease in the relative telomere length number with increasing passaging (P < 0.05). Isolated DPSCs highly expressed the CD markers: CD29, CD44, CD90, CD13, CD73 and CD166. In contrast, CD markers CD31, CD106, CD34 and CD45 were negative or low positive. We confirmed the high osteogenic and chondrogenic potential of the isolated stem cells. Isolated DPSCs did not show signs of cell degeneration or spontaneous differentiation during the entire cultivation. In addition, we were able to shorten the enzyme activity duration, and we were the first to demonstrate trypsin as the enzyme used for the enzymatic digestion method with the viability over 90 % of isolated DPSCs using this method.

OBJECTIVE

Determine the presence of mesenchymal stem cells (MSCs) in healthy periodontal tissue and periodontal granulation tissue (GT) and explore associations between immuno-regulatory molecules and selected subgingival microorganisms.

METHODS

Mesenchymal stem cells were isolated, propagated and characterised by flow cytometry from a region of healthy gingival tissue and inflamed GT of 10 systemically healthy non-smokers with chronic periodontitis. Tissue levels of immunoregulatory molecules were determined by qPCR and Gingival Crevicular Fluid (GCF) levels by ELISA. Subgingival plaque levels of periodontal pathogens were determined by qPCR RESULTS: Cells with MSC-properties were isolated from both inflamed GT and healthy gingival (G) tissue. A pro-inflammatory process predominated in GT which was partly reflected in GCF and putative periodontal pathogens were higher at diseased sites. However, there was no significant difference in surface levels of mesenchymal (CD90, CD73, CD146, CD271, STRO-1), endothelial (CD105, CD106), hematopoietic (CD34, CD45) and embryonic (SSEA-4) stem cell markers between MSCs isolated from GT and G tissue.

CONCLUSIONS

Periodontal lesions, albeit inflamed, retain healing potential as inferred by the presence of MSC-like cells with similar immunophenotypic characteristics to those found in healthy periodontal tissue. Therefore, there might be merits for healing in preserving sufficient GT in-situ during periodontal surgery.

Stem cells are most likely to solve all three of diabetes's problems at once, but the previous studies have mostly focused on bone marrow mesenchymal stem cells (MSCs) and adipose tissue-derived MSCs, and few studies have been done on pancreatic MSCs. In this study, pancreatic was collected to isolate MSCs from bovine, and then their biological characteristics such as growth kinetics, surface antigen, and multilineage potential were examined. Pancreatic MSCs of bovine (B-PMSCs) could be cultured for 65 passages in vitro. Growth kinetics analyses indicated that B-PMSCs had a strong capacity for self-renewal in vitro and their proliferation capacity appeared to decrease by passaging. Surface antigen detection showed that B-PMSCs expressed CD29, CD44, CD73, CD90, CD106, CD166, Vimentin, Nestin and Insulin, but not expressed CD34 and CD45. Furthermore, B-PMSCs could be induced to differentiate into adipocytes, osteoblasts and smooth muscle cells as indicated by reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. Most importantly, insulin-secreting cell differentiation of B-PMSCs exhibited islet-like clusters and dithizone staining displayed scarlet, and the response of the islet-like clusters to glucose suggested that high concentration glucose (20 mM) could quickly and persistently stimulate insulin release, and from the 2.0 h of the stimulation, the insulin of 20 mM glucose group were significantly higher than the 5.5 mM group. The B-PMSCs were isolated successfully, and the cells owned powerful self-renewal ability and multiple differentiative potential. Therefore, the present study plays an important role by providing a PMSCs choice for cell therapy of diabetes and tissue engineering.

Mesenchymal stem cells (MSCs) are able to self-renew and have multi-lineage differentiation potential. However, studies on ovine umbilical cord-derived MSCs (UC-MSCs) are limited. Our study aimed to isolate and characterize ovine UC-MSCs. We successfully isolated ovine UC-MSCs and defined their surface marker profile using immunofluorescence analysis. Ovine UC-MSCs were found to be positive for cell surface markers CD13, CD29, CD44, CD90, and CD106, and negative for cell surface marker CD45. Assessment of the proliferation potential of ovine UC-MSCs showed that from day 3 of cultivation a plateau phase was reached. And compare to passage 10, 15, 20 cells, passage 5 cells proliferating the fastest. Differentiation of ovine UC-MSCs into adipocytes, osteocytes, and chondrocytes was also demonstrated by staining for tissue-specific markers and using quantitative real-time polymerase chain reaction for specific marker gene expression. This study demonstrates the existence of a MSC population within the ovine umbilical cord, which maintained a normal karyotype up to passage 20.

Mesenchymal stem cells (MSCs) possess the ability to differentiate into multiple cell lineages, and thus, confer great potential for use in regenerative medicine and biotechnology. In this study, we attempted to isolate and characterize bovine tongue tissue-derived MSCs (boT-MSCs) and investigate the culture conditions required for long-term culturing of boT-MSCs. boT-MSCs were successfully isolated by the collagenase digestion method and their proliferative capacity was maintained for up to 20 or more passages. We observed a significant increase in the proliferation of boT-MSCs during the 20 consecutive passages under low glucose DMEM culture condition among the three culture conditions. These boT-MSCs presented pluripotency markers (Oct3/4 and Sox2) and cell surface markers, which included CD13, CD29, CD44, CD73, CD90, CD105, CD166, and MHC-I but not CD11b, CD14, CD31, CD34, CD45, CD80, CD86, CD106, CD117, and MHC-II at third passage. Moreover, these boT-MSCs could differentiate into mesodermal (adipocyte, osteocyte, and chondrocyte) cell lineages. Thus, this study suggests that the tongue of bovines could be used as a source of bovine MSCs.

OBJECTIVE

The expression of the cytokines IL-2, IL-6, IL-10, IFN-g and TNF-a and the adhesion proteins CD99 and CD106 was studied in the human testis at the protein level.

METHODS

The expression of the cytokines and the adhesion proteins was assessed using immunohistochemistry and immunoblotting.

RESULTS

None of the cytokines studied was present in the human testis, but CD99 and CD106 (VCAM-1) strongly were expressed in all the testes investigated. CD99 was present in the interstitial tissue of the human testis as well as in the Sertoli cells. The identity of the CD99+ interstitial cells is unclear. CD106 (VCAM-1) was present in Leydig cells as well as the basal parts of the Sertoli cells in the seminiferous tubules. In immunoblotting, CD99 was demonstrated at molecular ratios of 46-57 (kD). This is a novel isoform of the molecule.

CONCLUSIONS

The human testis produces both CD99 and CD106 and as CD106 mediates cell binding to lymphocytes, it is possible that the human Leydig cells adhere to lymphocytes like the rodent Leydig cells.

Mesenchymal stem cells (MSCs) in bone marrow and adipose tissues are expected to be effective tools for regenerative medicine to treat various diseases. To obtain MSCs that possess both high differentiation and tissue regenerative potential, it is necessary to establish an isolation system that does not require long-term culture. It has previously been reported that the cytoskeletal protein vimentin, expressed on the surfaces of multiple cell types, possesses N-acetylglucosamine- (GlcNAc-) binding activity. Therefore, we tried to exploit this interaction to efficiently isolate MSCs from rat bone marrow cells using GlcNAc-bearing polymer-coated dishes. Cells isolated by this method were identified as MSCs because they were CD34-, CD45-, and CD11b/c-negative and CD90-, CD29-, CD44-, CD54-, CD73-, and CD105-positive. Osteoblast, adipocyte, and chondrocyte differentiation was observed in these cells. In total, yields of rat MSCs were threefold to fourfold higher using GlcNAc-bearing polymer-coated dishes than yields using conventional tissue-culture dishes. Interestingly, MSCs isolated with GlcNAc-bearing polymer-coated dishes strongly expressed CD106, whereas those isolated with conventional tissue-culture dishes had low CD106 expression. Moreover, senescence-associated β-galactosidase activity in MSCs from GlcNAc-bearing polymer-coated dishes was lower than that in MSCs from tissue-culture dishes. These results establish an improved isolation method for high-quality MSCs.

Studies have shown that high-fat diets cause blood vessel damage, however, assessing pathological effects accurately and efficiently is difficult. In this study, we measured particle levels of static endothelium (CD31+ and CD105+) and activated endothelium (CD62E+, CD54+ and CD106+) in plasma. We determined individual responses to two dietary regimens in two groups of baboons. One group (n = 10), was fed a diet high in simple carbohydrates and saturated fats (the HSF diet) and the other (n = 8) received a diet high in simple carbohydrates and unsaturated fats (the HUF diet). Plasma samples were collected at 0, 3, and 7 weeks. The percentages of CD31+ and CD62E+ particles were elevated at 3 weeks in animals fed either diet, but these elevations were statistically significant only in animals fed the HUF diet. Surprisingly, both percentages and counts of CD31+ particles were significantly lower at week 7 compared to week 0 and 3 in the HSF group. The median absolute counts of CD105+ particles were progressively elevated over time in the HSF group with a significant increase from week 0 to 7; the pattern was somewhat different for the HUF group with significant increase from week 3 to 7. The counts of CD54+ particles exhibited wide variation in both groups during the dietary challenge, while the median counts of CD106+ particles were significantly lower at week 3 than at week 0 and week 7. Endothelial particles exhibited time-dependent changes, suggesting they were behaving as quantifiable surrogates for the early detection of vascular damage caused by dietary factors.

The antifibrotic properties of spiperone and its effect on stem and progenitor cells were studied on the model of reversible bleomycin-induced pulmonary fibrosis in C57Bl/6 mice. Spiperone reduced infiltration of the alveolar interstitium and alveolar ducts with inflammatory cells and prevented the growth of the connective tissue in the parenchyma of bleomycin lungs. Apart from anti-inflammatory effect, spiperone suppressed bone marrow hemopoietic cells (CD3, CD45R (B220), Ly6C, Ly6G (Gr1), CD11b (Mac1), TER-119)-, Sca-1+, c-Kit+, CD34- and progenitor hemopoietic cells (granulocyte-erythroid-macrophage-megakaryocytic and granulocyte CFU). Spiperone-induced disturbances of fi brogenesis were paralleled by restoration of endothelial cells in the lung parenchyma, reduction of the number of circulating bone marrow cells and lung mesenchymopoietic cells (mesenchymal multipotent stromal cells (CD31-, CD34-, CD45-, CD44+, CD73+, CD90+, CD106+) and progenitor fi broblast cells), and suppression of multilineage differentiation of multipotent mesenchymal stromal cells (including fi broblast-lineage cells).

Mesenchymal stem cells (MSCs) have the ability to migrate to the site of injury or inflammation, and to contribute to the healing process. Since patients treated with MSCs are often users of analgesic drugs, to relieve their uncomfortable pain associated with the tissue disorder, there is a possibility of negative effects of these drugs on the migration of endogenous and exogenous MSCs. Therefore, we tested the impact of acute and chronic treatment with morphine on the migration and organ distribution of exogenous adipose tissue-derived MSCs in mouse models. Firstly, we showed that the incubation of MSCs with morphine significantly reduced the expression of adhesive molecules CD44 (HCAM), CD54 (ICAM-1) and CD106 (VCAM-1) on MSCs. Using a model of systemic administration of MSCs labeled with vital dye PKH26 and by the application of flow cytometry to detect living CD45^-PKH26⁺ cells, we found a decreased number of labeled MSCs in the lung, spleen and bone marrow, and a significantly increased number of MSCs in the liver of morphine-treated recipients. A skin allograft model was used to study the effects of morphine on the migration of exogenous MSCs to the superficial wound. Intraperitoneally administered MSCs migrated preferentially to the wound site, and this migration was significantly decreased in the morphine-treated recipients. The present results showed that morphine significantly influences the distribution of exogenous MSCs in the body, and decreases their migration to the site of injury.

Keywords: Acute and chronic morphine treatment; Adhesive molecules; Cell migration; Mesenchymal stem cells; Skin graft model.

Mechanical forces, hypoxia, and oxidative stress contribute to skin renewal, perfusion, and wound healing, but how are they regulating subcutaneous adipose-derived stem cells (ASCs) in the inflammatory microenvironment associated to skin repair and disorders is unknown. In this study, ASCs were isolated from lipoaspirate samples from plastic surgery patients, primary cultured and their differentiation and secretion of a panel of cytokines with pronounced effects on skin repair and angiogenesis were studied under mechanical stimulation by intermittent fluid flow, 1% hypoxia and oxidative stress by glutathione (GSH) depletion with buthionine sulfoximine (BSO) treatment. Mechanical action of fluid flow did not alter mesenchymal phenotype of CD90+ /CD29+ /CD44+ /CD34- /CD106- /CD45- ASCs; however, it remarkably induced ASC secretion of human umbilical vein endothelial cell (HUVEC) migration-stimulating factors. Multiplex Luminex assay further confirmed an increased secretion of VEGF, G-CSF, HGF, Leptin, IL-8, PDGF-BB, Angiopoietin-2, and Follistatin from mechanically-stimulated ASCs via cyclooxygenase-2. Consistent with this mechanism, GSH depletion and hypoxia also increased ASC secretion of VEGF, IL-8, leptin, Angiopoitein-2, and PDGF-BB. However, mechanical action of fluid flow abrogated VEGF and HUVEC migration-stimulating activity from GSH-depleted and hypoxic ASCs. Conversely, GSH depletion and hypoxia abrogated VEGF and HUVEC migration-stimulating activity from mechano-stimulated ASCs. Although mechanical action of fluid flow, hypoxia, and GSH-depletion had independent proangiogenic-stimulating activity on ASCs, mechanical stimulation had opposite effects on proangiogenic factor secretion from ASCs with and without oxidative stress. These data uncover the role of hypoxia and endogenous redox balance during the proangiogenic response of ASCs and other mesenchymal-derived cell types to mechanical action of interstitial fluid flow. J. Cell. Physiol. 232: 2158-2167, 2017. © 2016 Wiley Periodicals, Inc.

Skeletal homeostasis is dynamically influenced by the immune system. Low density lipoprotein receptor-related protein-5 (LRP5) is a co-receptor of the Wnt signaling pathway, which modulates bone metabolism in humans and mice. Immune disorders can lead to abnormal bone metabolism. It is unclear whether and how LRP5 alters the balance of the immune system to modulate bone homeostasis. In this study, we used primary osteoblast to detect the differentiation of osteoblasts in vitro, the immune cells of spleen and bone marrow of 6-month old LRP5 heterozygote (HZ) and wild-type (WT) mice were analyzed by Flow cytometry. We found that LRP5+/- could influence the differentiation of osteoblasts by decreasing the mRNA level of Osterix, and increasing the mRNA level of Runx2 and the ratio of receptor activator for nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG). In the LRP5+/- mice, percentages of NK cells, CD3e+ cells, and CD8a+ T cells were increased in both spleen and bone marrow, and percentages of CD106+ cells and CD11c+ cells were increased in spleen while decreased in bone marrow, conversely, CD62L+ cells were decreased in spleen while increased in bone marrow compared to the WT mice. Percentages of CD4+ cells, CD14+ cells, and CD254+ cells were increased in the spleen, and CTLA4+ cells were increased in the bone marrow of the LRP5+/- mice. The mRNA level of Wnt signaling molecules such as β-catenin, and c-myc were decreased and APC was increased in spleen lymphocytes and bone marrow lymphocytes, and the mRNA level of Wnt3a was decreased in spleen lymphocytes while no change in bone marrow lymphocytes was seen with silencing LRP5 by specific small interfering RNA. In conclusion, heterozygous deletion of the LRP5 gene in mice could alter the profile of the immune cells, influence the balance of immune environment, and modulate bone homeostasis, which might present a potential mechanism to explore the Wnt signaling pathway in the modulation of the immune system.

Meniscus injuries are among the most common orthopedic injuries. Tears in the inner one-third of the meniscus heal poorly and present a significant clinical challenge. In this study, we hypothesized that progenitor cells from healthy human articular cartilage (chondroprogenitor cells [C-PCs]) may be more suitable than bone-marrow mesenchymal stem cells (BM-MSCs) to mediate bridging and reintegration of fibrocartilage tissue tears in meniscus. C-PCs were isolated from healthy human articular cartilage based on their expression of mesenchymal stem/progenitor marker activated leukocyte cell adhesion molecule (ALCAM) (CD166). Our findings revealed that healthy human C-PCs are CD166+, CD90+, CD54+, CD106- cells with multilineage differentiation potential, and elevated basal expression of chondrogenesis marker SOX-9. We show that, similar to BM-MSCs, C-PCs are responsive to the chemokine stromal cell-derived factor-1 (SDF-1) and they can successfully migrate to the area of meniscal tissue damage promoting collagen bridging across inner meniscal tears. In contrast to BM-MSCs, C-PCs maintained reduced expression of cellular hypertrophy marker collagen X in monolayer culture and in an explant organ culture model of meniscus repair. Treatment of C-PCs with SDF-1/CXCR4 pathway inhibitor AMD3100 disrupted cell localization to area of injury and prevented meniscus tissue bridging thereby indicating that the SDF-1/CXCR4 axis is an important mediator of this repair process. This study suggests that C-PCs from healthy human cartilage may potentially be a useful tool for fibrocartilage tissue repair/regeneration because they resist cellular hypertrophy and mobilize in response to chemokine signaling. Stem Cells 2018.

Background: Mesenchymal stromal cells (MSCs), multipotent progenitors that can be isolated from a variety of different tissues, are becoming increasingly important as cell therapeutics targeting immunopathologies and tissue regeneration. Current protocols for MSC isolation from bone marrow (BM) rely on density gradient centrifugation (DGC), and the production of sufficient MSC doses is a critical factor for conducting clinical MSC trials. Previously, a Good Manufacturing Practice (GMP)-compatible non-woven fabric filter device system to isolate MSCs was developed to increase the MSC yield from the BM. The aim of our study was to compare high-resolution phenotypic and functional characteristics of BM-MSCs isolated with this device and with standard DGC technology.

Methods: Human BM samples from 5 donors were analyzed. Each sample was divided equally, processing by DGC, and with the filter device. Stem cell content was assessed by quantification of colony-forming units fibroblasts (CFU-F). Immunophenotype was analyzed by multicolor flow cytometry. In vitro trilineage differentiation potential, trophic factors, and IDO-1 production were assessed. Functionally, immunomodulatory potential, wound healing, and angiogenesis were assayed in vitro.

Results: The CFU-F yield was 15-fold higher in the MSC preparations isolated with the device compared to those isolated by DGC. Consequently, the MSC yield that could be manufactured at passage 3 per mL collected BM was more than 10 times higher in the device group compared to DGC (1.65 × 10⁹ vs. 1.45 × 10⁸). The immunomodulatory potential and IDO-1 production showed donor-to-donor variabilities without differences between fabric filter-isolated and DGC-isolated MSCs. The results from the wound closure assays, the tube formation assays, and the trilineage differentiation assays were similar between the groups with respect to the isolation method. Sixty-four MSC subpopulations could be quantified with CD140a⁺CD119⁺CD146⁺ as most common phenotype group, and CD140a⁺CD119⁺CD146⁺MSCA-1^-CD106^-CD271^- and CD140a⁺CD119⁺CD146^-MSCA-1^-CD106^-CD271^- as most frequent MSC subpopulations. As trophic factors hepatocyte growth factor, epidermal growth factor, brain-derived neurotrophic factor, angiopoietin-1, and vascular endothelial growth factor A could be detected in both groups with considerable variability between donors, but independent of the respective MSC isolation technique.

Conclusion: The isolation of MSCs using a GMP-compatible fabric filter system device resulted in higher yield of CFU-F, producing substantially more MSCs with similar subpopulation composition and functional characteristics as MSCs isolated by DGC.

Keywords: GMP; function; heterogeneity; mesenchymal stromal cell; production; subpopulation.

Mouse embryonic fibroblasts (MEFs) accessibility coupled with their simple generation make them as a typical embryonic cell model and feeder layer for in vitro expansion of pluripotent stem cells (PSCs). In this study, a mechanical isolation technique was adopted to isolate MEFs and the efficiency of this technique was compared with enzymatic digestion method. The suspended MEFs were prepared either by mechanical method or 0.25% trypsin enzymatic digestion. The effect of tissue processing on cell apoptosis/necrosis, morphology, viable cell yield, population doubling time, surface marker expression, and the capacity to support PSCs were determined. The mechanical method yielded a significantly higher number of viable cells. However, it showed similar morphology and proliferation characteristics as compared to enzymatic digestion. The mechanical method induced slight apoptosis in MEFs; however, it did not exert the necrotic effect of trypsinization. Treatment of tissue slurry with trypsin solution caused cell lysis and subsequently cell clump formation. Mechanically isolated cells exhibited a higher expression of the MEF surface antigens Sca1, CD106, and CD105. The PSCs on mechanically isolated MEFs displayed a higher expression of pluripotency genes, and formed more compact colonies with a stronger tendency to crowding compared with those cultured on cells isolated by enzymatic digestion. The mechanical method based on tissue inter-syringe processing is relatively a rapid and simple method for MEF isolation. Compared to the enzymatic digestion, the cells obtained from this method show higher expression of embryonic fibroblasts markers and a more functional capacity in supporting PSCs culture.

Keywords: Cell proliferation; Cytological techniques; Embryonic stem cells; Feeder layer; Fibroblasts; Primary cell culture.

Objective: To investigation the types and frequencies of thalassemia gene mutations in pregnant population in Nanping area of Fujian Province, so as to provide a basis for prevention and control of birth children with moderate and severe thalassaemia in this area.

Methods: The genotyping of α and β thalassemia was performed using the gap-PCR (gap-PCR) technique combined with reverse dot blot (RDB). The genotyping test was performed by Gap-PCR for three rare deficient thalassemia. The cases with negative detection were further detected by Sanger sequencing method, so as to identify rare α or β thalassemia mutation.

Results: 1120 specimens were genotyped for thalassemia, out of them 547 thalassemia genes were determined. The detection rate was 48.8% (547/1120). 340 specimens were diagnosed as α thalassemia, and the detection rate was 30.6%, including 266 cases of --^SEA/αα, 44 cases of -α^3.7/αα, 12 cases of -α^4.2/αα, 8 cases of αα^QS/αα,. 3 cases of Hb H disease ( 2 cases of --^SEA/-α^3.7, 1 case of --^SEA/-α^4.2), 2 cases of αα^CS/αα, 2 cases of αα^WS/αα, 1 case of -α^3.7/-α^3.7, and 1 case of -α^3.7/αα^QS. Also, they contain 11 cases of rare α thalassemia, 8 kinds of rare types of α thalassemia mutations in combination, such as 4 cases of αα^{IVS-II-55 (T→G) in α1}/αα, 1 case of αα^{IVS-I-62 (C→T) in α1}/αα, 1 case of αα^{CD106（CTG→GTG）in α2}/αα, 1 case of αα^{HBA2:c.-24C>G}/αα, 1 case of αα^{IVS-II-55 (T→G) in α1}/αα^{IVS-II-55 (T→G) in α1}, 1 case of αα^{IVS-II-55 (T→G) in α1}/αα^{IVS-II-119 (G;+CTCGGCCC) in α2}, 1 case of αα^{IVS-II-88 (G→A) in α2}/αα, and 1 case of --^THAI/αα. Among them, 5 α mutation sites were first reported, namely αα^{IVS-I-62 (C→T) in α1}, αα^{IVS-II-55 (T→G) in α1}, αα^{IVS-II-119 (G; +CTCGGCCC ) in α2}, αα^{IVS-II-88 (G→A) in α2} and αα^{CD106 (CTG→GTG) in α2}; 2 α thalassemia mutation sites: αα^{HBA2: c.-24C>G} and --^THAI were detected again in the Chinese population, respectively. 188 specimens were diagnosed as β thalassemia with a detection rate of 16.8%. Among them, 68 cases of β^IVS-II-654/βN, 47 cases of β^CD41-42/βN, 20 cases of β^CD17/βN, 17 cases of β^-28/βN, 7 cases of β^CD27-28/βN, 7 cases of βE/βN, 3 cases of β^CD71-72/βN and 2 cases of β^CD43/βN. And 17 cases were diagnosed as rare β thalassemia, 8 kinds of rare types were β thalassemia mutations in combination. There were 4 cases of β^{IVS-II-81 (C→T)}/βN, 3 cases of β^{Hb J-Bangkok}/βN, 3 cases of β^{Hb New York}/βN, 2 cases of β^{-96 (G→T)}/βN, 2 cases of β^{IVS-II-806 (G→C)}/βN, 1 case of β^{Codons 8/9}/βN, 1 case of β^{Hb G-Coushatta}/βN, 1 case of β^{IVS-II-827 (A→T)}/βN. Among them, 3 β thalassemia mutation sites were reported for the first time, namely β^{-96 (G→T)}, β^{IVS-II-806 (G→C)} and β^{IVS-II-827 (A→T)}; it was found that in the Chinese population as β^{Codons 8/9}, β^{Hb G-Coushatta}, β^{Hb J-Bangkok}, β^{Hb New York}, and β^{IVS-II-81 (C→T)}, respectively. 19 cases were diagnosed as αβ-complex thalassemia, out of which 15 types of thalassemia mutation combinations were detected. They contain 2 cases of rare αβ-complex thalassemia, which are αα^{IVS-II-55 (T→G)}/αα complex β^{IVS-II-81 (C→T)}/βN, αα^{IVS-II-65 (G→A)} in α1/αα complex β^{Hb G-Coushatta}/βN.

Conclusion: The types of thalassemia gene mutations in Nanping area of Fujian province are genetically heterogeneous. The prevention and control strategies of thalassaemia in this area should be based on the prevention and treatment of common α thalassemia and β thalassaemia. And the attention should be paid to the types of rare and unknown gene mutations using screening and testing method.

题目: 中国福建南平地区地中海贫血基因突变类型研究.

目的: 了解福建省南平地区地中海贫血基因突变类型及频率，为本地区预防和控制中型和重型地中海贫血患儿的出生提供依据，减少出生缺陷.

方法: 采用跨越断裂点PCR（gap-PCR）技术结合反向点杂交法（RDB）进行α和β地中海贫血基因分型检测，对基因分型检测阴性者应用Gap-PCR法进行3种罕见缺失型地中海贫血检测，对检测阴性者采用Sanger测序法进行罕见α地中海贫血突变和β地中海贫血突变检测.

结果: 1 120例标本参加地中海贫血基因分型检测，检出547例地中海贫血基因，检出率为48.8%（547/1120）。340例标本确诊为α地中海贫血，检出率为30.6%，其中--^SEA/αα 256例、-α^3.7/αα 44例、-α^4.2/αα 12例、αα^QS/αα 8例、Hb H病3例（--^SEA/-α^3.7 2例，--^SEA/-α^4.2 1例）、αα^CS/αα 2例、αα^WS/αα 2例、-α^3.7/-α^3.7 1例、-α^3.7/αα^QS 1例。1 120例中含有11例罕见型α地中海贫血，8种罕见α地中海贫血突变组合类型，分别是αα^{IVS-II-55 (T→G) in α1}/ αα 4例、αα^{IVS-I-62(C→T) in α1}/αα 1例、αα^{CD106（CTG→GTG）in α2}/αα 1例、αα^{HBA2: c.-24C>G}/αα 1例、αα^{IVS-II-55(T→G) in α1}/ αα^{IVS-II-55(T→G) in α1}IVS-II-55(T→G) in α1/αα^{IVS-II-119(G; +CTCGGCCC) in α2} 1例、αα^{IVS-II-88(G→A) in α2}/αα 1例和--^THAI/αα 1例。其中，5种α突变位点是首次报道，分别是αα^{IVS-I-62(C→T) in α1}、αα^{IVS-II-55(T→G) in α1}、αα^{IVS-II-119(G; +CTCGGCCC) in α2}、αα^{IVS-II-88(G→A) in α2}和αα^{CD106（CTG→GTG）in α2}；2种α突变位点再次在中国人群中检测到，分别是αα^{HBA2:c.-24C>G}和--^THAI。188例标本确诊为β地中海贫血，检出率为16.8%，其中β^IVS-II-654/βN 68例、β^CD41-42/βN 47例、β^CD17/βN 20例、β^-28/βN 17例，β^CD27-28/βN 7例、βE/βN 7例、β^CD71-72/βN 3例、β^CD43/βN 2例。同时，含有17例罕见型β地中海贫血，8种罕见β地中海贫血突变组合类型，分别是β^{IVS-II-81(C→T)}/βN 4例、β^{Hb J-Bangkok}/βN 3例，β^{Hb New York}/βN 3例、β^-96(G→T)/βN 2例、β^{IVS-II-806(G→C)}/βN 2例、β^{Codons 8/9}/βN 1例、β^{Hb G-Coushatta}/βN 1例、β^{IVS-II-827(A→T)}/βN 1例。其中有3个β突变位点是首次报道，分别是β^-96(G→T)、β^{IVS-II-806(G→C)}和β^{IVS-II-827(A→T)}；β突变位点再次在中国人群中检测到，分别是β^{Codons 8/9}、β^{Hb G-Coushatta}、β^{Hb J-Bangkok}、β^{Hb New York}和β^{IVS-II-81(C→T)}。19例确诊α地中海贫血复合β地中海贫血，共检出15种地中海贫血突变组合类型，包含2例罕见稀有α地中海贫血复合β地中海贫血，分别是αα^{IVS-II-55(T→G)}/αα复合β^{IVS-II-81(C→T)}/βN、αα^{IVS-II-65(G→A)in α1}/αα复合β^{Hb G-Coushatta}/βN.

结论: 福建省南平地区地中海贫血基因突变类型具有遗传异质性，本地区地中海贫血防治策略应该以常见α-地中海贫血和β-地中海贫血的防治干预为主，同时也要重视罕见和未知基因突变类型的筛查和检测.

The experimental aim of this study was to determine, in vitro, the effects of glucose-induced EMPs on endothelial cell expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 and platelet cell adhesion molecule-1 (PECAM-1). Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 10⁵ cells/condition. HUVECs were incubated with media containing either 25 mM d-glucose (concentration representing a hyperglycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144⁺) and concentration were determined by flow cytometry. HUVECs (3 × 10⁶ cells/condition) were treated with either high glucose-derived EMPs (hgEMPs) or normal glucose-derived (ngEMPs) for 24 h and surface expression of E-selectin (CD62E-PE), ICAM-1 (CD54-FITC), VCAM-1 (CD106-APC) and PECAM-1 (CD31-BV) was assessed by flow cytometry and reported as mean fluorescent intensity (MFI). Hyperglycemic-derived EMPs induced significantly higher surface expression of E-selectin (2614 ± 132 vs. 2010 ± 204 MFI), ICAM-1 (2110 ± 81 vs. 1688 ± 152 MFI), VCAM-1 (3589 ± 431 vs. 2134 ± 386) and PECAM-1 (4237 ± 395 vs. 2525 ± 269 MFI) on endothelial cells than EMPs from normoglycemic conditions. Microparticle-induced cell adhesion molecule expression provides potential novel mechanistic insight regarding the accelerated risk of atherosclerotic vascular disease associated with hyperglycemia.

Manufacturing processes for autologous cell therapy need to reproducibly generate in specification (quality and quantity) clinical product. However, patient variability prevents the level of control of cell input material that could be achieved in a cell line or allogeneic-based process. We have applied literature data on bone marrow-derived mesenchymal stromal cells variability to estimate probability distributions for stem cell yields given underlying truncated normal distributions in total nucleated cell concentration, stem cell percentage and plausible aspirate volumes. Monte Carlo simulation identified potential variability in harvested stem cell number in excess of an order of magnitude. The source material variability was used to identify the proportion of donor manufacturing runs that would achieve a target yield specification of 2E7 cells in a fixed time window with given proliferative rates and different aspirate volumes. A rapid, screening, development approach was undertaken to assess culture materials and process parameters (T-flask surface, medium, feed schedule) to specify a protocol with identified proliferative rate and a consequent model-based target aspirate volume. Finally, four engineering runs of the candidate process were conducted and a range of relevant quality parameters measured including expression of markers CD105, CD73, CD44, CD45, CD34, CD11b, CD19, HLA-DR, CD146 (melanoma cell adhesion molecule), CD106 (vascular cell adhesion molecule) and SSEA-4, specific metabolic activity and vascular endothelial growth factor secretion, and osteogenic differentiation potential. Our approach of using estimated distributions from publicly available information provides a route for data-poor earl- stage developers to plan manufacture with defined risk based on rational assumptions; furthermore, the models produced by such assumptions can be used to evaluate candidate processes, and can be incrementally improved with accumulating distribution understanding or subdivision by new process variables.

Migration of leukocytes to inflammation sites through vascular endothelium is controlled by the interactions of adhesion molecules expressed on both endothelial cells and leukocytes, most of which are already covered by cluster of differentiation (CD) codes. We examined the expression of a variety of endothelial cell adhesion molecules in human dental pulp vasculature to obtain further evidence on the tissue distribution and function of these molecules by using an indirect immunoperoxidase technique. We obtained the pulp tissue samples from teeth extracted due to orthodontic reasons as controls and compared with those extracted due to chronic periodontitis. In all samples, both CD31 and CD146 were expressed by arterial, venous, and capillary endothelia. There was no significant difference between the staining intensity of normal and inflamed pulp tissues. CD102 expression on the endothelium was significantly stronger in chronic periodontitis pulp samples. CD106, CD62-E, CD62-P, CD105, and CD54 were variably expressed in control and chronic periodontitis groups. Our results indicate that CD102 represents the major endothelial cell adhesion molecule probably involved in the inflammatory reactions in chronic periodontitis.

Very small embryonic-like stem cells (VSELs) represent a unique rare population of adult stem cells (SCs) sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM-) derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I) antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog) on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS), we also optimized sorting protocol for separation of CD45(-)/Lin(-)/CD106(+) rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury.

Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4+ T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4+ T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4+ T cells significantly enhanced IL-9 production by CD4+ T cells. Transwell experiments suggested that UC-MSC promotion of IL-9 production by CD4+ T cells was dependent on cell-cell contact. Upregulated expression of CD106 was observed in UC-MSC co-cultured with CD4+ T cells, and the addition of a blocking antibody of CD106 significantly impaired the ability of UC-MSC to promote IL-9 production by CD4+ T cells. Therefore, the results of the present study demonstrated that UC-MSC promoted the generation of IL-9 producing cells, which may be mediated, in part by CD106. The findings may act to expand understanding and knowledge of the immune modulatory role of UC-MSC.

Combination of stem cell technology and 3D biofabrication approaches provides physiological similarity to in vivo tissues and the capability of repairing and regenerating damaged human tissues. Mesenchymal stem cells (MSCs) have been widely used for regenerative medicine applications because of their immunosuppressive properties and multipotent potentials. To obtain large amount of high-quality MSCs without patient donation and invasive procedures, we differentiated MSCs from human-induced pluripotent stem cells (hiPSC-MSCs) using serum-free E6 media supplemented with only one growth factor (bFGF) and two small molecules (SB431542 and CHIR99021). The differentiated cells showed a high expression of common MSC-specific surface markers (CD90, CD73, CD105, CD106, CD146, and CD166) and a high potency for osteogenic and chondrogenic differentiation. With these cells, we have been able to manufacture MSC tissue rings with high consistency and robustness in pluronic-coated reusable PDMS devices. The MSC tissue rings were characterized based on inner diameter and outer ring diameter and observed cell-type-dependent tissue contraction induced by cell-matrix interaction. Our approach of simplified hiPSC-MSC differentiation, modular fabrication procedure, and serum-free culture conditions has a great potential for scalable manufacturing of MSC tissue rings for different regenerative medicine applications.

OBJECTIVE

Circulating endothelial cells (CECs) have been described in different conditions with vascular injury. Vascular abnormalities play a key role in the pathogenesis of Systemic Sclerosis (SSc). The aim of our study was to look for the presence of CECs in SSc patients and to evaluate their clinical significance.

METHODS

We studied 52 SSc patients and 40 healthy controls (HC). Five-parameter, 3-color flow cytometry was performed with a FACScan. CECs were defined as CD45 negative, CD31 and P1H12 positive, and activated CECs as CD45 negative and P1H12, CD62, or CD106 positive.

RESULTS

Total and activated CEC counts were significantly higher in SSc patients when compared with HC and positively correlated with disease activity score. We found a significant association between CECs and disease activity; as regard with organ involvement, CEC number correlate with the severity of pulmonary hypertension.

CONCLUSIONS

Raised counts of CECs may represent direct evidence of active vascular disease in SSc as regard as visceral involvement, the association between CECs and pulmonary hypertension suggest a relevant role for CECs as a marker of prominent endothelial involvement.