Immunophenotypic evaluation,and physiological and laboratory correlations of hematopoietic stem cells from umbilical cord blood

The use of umbilical cord blood stem cells is an efficient alternative for the transplantation of hematopoietic progenitor cells. A number of factors can influence the volume and amount of CD34⁺ cells, which are considered as immature and capable of proliferation. Quantification of CD34⁺ cells, evaluation of CD38 and c-kit molecules on these cells, as well as correlations of such factors as maternal age, gestational age, newborn sex and weight, umbilical cord length, placental weight with increased volume and concentration of immature cells, among others, were performed in 70 blood samples from term newborns. The mean volume of umbilical cord blood collected was 53.8±33.6 mL, where 30.96±18.9 CD34⁺/µL UCB cells were found, of which 16.66±8.32% were CD34⁺ CD38- cells, and 47.23±24.0% were CD34⁺ CD117^- cells. Newborn weight and placental weight were positively correlated with increased volume of collected UCB. The volume of collected blood was found to affect the absolute count of CD34⁺ cells and the relative value of these among total nucleated cells, as well as the percentage of CD34⁺CD117⁺ and CD34⁺CD117^- cells. CD34⁺ cells were positively correlated with leukocytes, and gestational age was negatively correlated with the number of CD34⁺ cells. Our results confirm the importance of the accurate quantification of CD34⁺ cells and their subsets, and that many factors may be related to the higher number of hematopoietic stem cells, which are crucial for successful transplantation.     

CD34⁺ CD38^- subpopulation without CD123 and CD44 is responsible for LSC and correlated with imbalance of immune cell subsets in AML

Acute myeloid leukemia (AML) is regarded as a stem cell disease. However, no one unique marker is expressed on leukemia stem cells (LSC) but not on leukemic blasts nor normal hematopoietic stem cells (HSC). CD34⁺ CD38- with or without CD123 or CD44 subpopulations are immunophenotypically defined as putative LSC fractions in AML. Nevertheless, markers that can be effectively and simply held responsible for the intrinsical heterogeneity of LSC is still unclear. In the present study, we examined the frequency of three different LSC subtypes (CD34⁺ CD38^-, CD34⁺ CD38^- CD123⁺ , CD34⁺ CD38^- CD44⁺ ) in AML at diagnosis. We then validated their prognostic significance on the relevance of spectral features for diagnostic stratification, immune status, induction therapy response, treatment effect maintenance, and long-term survival. In our findings, high proportions of the above three different LSC subtypes were all significantly characterized with low complete remission (CR) rate, high relapse/refractory rate, poor overall survival (OS), frequent FLT3-ITD mutation, the high level of regulatory T cells (Treg) and monocytic myeloid-derived suppressor cells (M-MDSC). However, there was no significant statistical difference in all kinds of other clinical performance among the three different LSC groups. It was demonstrated that CD34⁺ CD38^- subpopulation without CD123 and CD44 might be held responsible for LSC and correlated with an imbalance of immune cell subsets in AML.     

Calcitonin gene related peptide modified mesenchymal stem cells reduce restenosis after carotid balloon injury in rats

This work aimed to investigate the effects of calcitonin gene-related peptide (CGRP)-modified mesenchymal stem cells (MSCs) on vascular stenosis in carotid balloon-injured rats. The CGRP gene labeled with EGFP was transfected into bone marrow MSCs, and CGRP protein expression in MSCs was confirmed by immunofluorescence assays. A rat carotid balloon injury model was established using a surgical method. CGRP-modified MSCs were orthotopically transplanted into the injured area of the rats. At 28 days after cell transplantation, EGFP and CD31 expression was detected by immunofluorescence staining. Hematoxylin-eosin (H&E) staining was used to detect the intima/media area of the injured carotid artery stenosis site, and the expression of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. MSCs from rat bone marrow positively expressed CD29 and negatively expressed CD45. In vivo immunofluorescence staining showed that EGFP expression was significantly increased at the vascular injury site of rats transplanted with MSC_CGRP compared with the control group on the 28th day after cell transplantation and that the damaged vessels exhibited continuous CD31 expression. H&E staining showed that the vascular intimal proliferation area of rats transplanted with MSC_CGRP was significantly reduced compared with that of other groups. Furthermore, the immunohistochemistry results showed that PCNA expression in the endothelium of the MSC_CGRP group was lower than that of the other groups. Therefore, MSCs transfected with the CGRP gene can express the CGRP protein, and the implantation of CGRP-modified MSCs at the damaged site after carotid balloon-induced injury can reduce the neointimal area.     

Immunophenotypic,immunocytochemistry, ultrastructural,and cytogenetic characterization of mesenchymal stem cells from equine bone marrow

The aim of this study was to isolate, culture, and characterize mesenchymal stem cells (MSCs) from horse bone marrow (BM) using the techniques of flow cytometry, immunocytochemistry, cytogenetics, and electron microscopy. Immunophenotypic analysis revealed the presence of MSCs with high expression of the CD90 marker, lower expression of the CD44 marker, and absent expression of the CD34 marker. In assays of differentiation, the positive response to osteogenic (OST), chondrogenic (CDG), and adipogenic (ADP) differentiation signals was observed and characterized by deposition of calcium‐rich extracellular matrix (OST), proteoglycans and collagen II (CDG) and intracellular deposition of fat drops (ADP). In immunocytochemical characterization, MSCs were immunopositive for CD44, vimentin, and PCNA, and they were negative for CD13. In the ultrastructural analysis of MSCs, the most outstanding characteristic was the presence of rough endoplasmic reticulum with very dilated cisterns filled with a low electrodensity material. Additionally, MSCs had normal karyotypes (2n = 64) as evidenced by cytogenetic analysis, and aneuploidy in metaphase was not observed. The protocols for isolating, culturing, and characterizing equine MSCs used in this study were shown to be appropriate for the production of a cell population with a good potential for differentiation and without aneuploidy that can be used to study future cellular therapies. Microsc. Res. Tech. 76:618–624, 2013. © 2013 Wiley Periodicals, Inc.     

Berberine inhibits the proliferation of pancreatic cancer cells by targeting pancreatic cancer stem cells through regulating EMT signaling pathway

Pancreatic ductal adenocarcinoma (PDAC) is universally acknowledged as the cancer with the highest mortality rate. Berberine has high medicinal value and has been used as an anti-cancer agent. Hence the purpose of this study was to investigate the anti-cancer effect of berberine in PDAC. Berberine was shown to have a selective anti-cancer effect on PDAC by MTT assay in vitro. Pancreatic cancer stem cells (PCSCs), regulated by epithelial–mesenchymal transition (EMT), could promote the proliferation of PDAC cells. However, berberine suppressed the proliferation and stemness of PCSCs through immunofluorescence staining, stem cell sphere assays and so forth in vitro. In vivo, berberine reduced tumor size and decreased the expression levels of Ki67, a marker of cellular proliferation, in orthotopic pancreatic tumors. In addition, berberine inhibited the EMT signaling pathway by RT-PCR and Western blotting methods both in vitro and in vivo. Our study indicates that berberine inhibits the proliferation of PDAC cells both in vivo and in vitro. The mechanism of the anti-cancer effect of berberine likely involves the inhibition of EMT. Therefore, berberine may be a novel antineoplastic drug with clinical efficacy in PDAC.     

Hematopoietic progenitor constituents and adherent cell progenitor morphology isolated from black-rumped agouti (Dasyprocta prymnolopha, Wagler 1831) bone marrow

Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black‐rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10⁷ cells (ACP). Thirteen 25‐cm² culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10⁶ cells per milliliter, and placed in liquid nitrogen at ?196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

一种新的提取脂肪间充质干细胞的方法及对提取细胞的分离、培养及鉴定

目的：探讨建立一种新的从膨胀液中提取脂肪间充质干细胞（ADSCs）的分离方法。方法：收集含有脂肪间充质干细胞的膨胀液,然后从膨胀液中分离出脂肪间充质干细胞并进行体外培养,观察培养间充质干细胞生长状态,流式细胞术检测间充质干细胞干性标记物,细胞生长曲线比较新方法与运用传统方法分离脂肪间充质干细胞的增殖活性,多向诱导分化鉴定其向成骨,成软骨及成脂方向分化的能力。结果：成功建立了一种新的从膨胀液中提取脂肪间充质干细胞的分离方法;分离自膨胀液的间充质干细胞数量虽然低于与等体积脂肪组织来源的间充质干细胞,但细胞生长曲线分析结果表明其增殖速度快,生长至第8天时,密度基本等同于脂肪组织来源间充质干细胞。间充质干细胞表面分子标记物CD73,CD90,CD105,CD45,CD34,CD11b,CD19,HLA—DR表达测定结果显示正常,阳性细胞率与脂肪组织来源的干细胞相近。多向诱导分化结果显示从膨胀液中分离的脂肪间充质干细胞可以向成脂、成骨和成软骨三向分化。结论：新方法分离的细胞确为脂肪间充质干细胞,符合国际干细胞协会规定的定义标准。     

Synchrotron radiation X‐ray micro‐fluorescence: Protocol to study mesenchymal stem cells

The micro‐X‐ray fluorescence by synchrotron radiation (μ‐XRF) is a method to determine the composition of tissues without destroying the samples. However, this technique has never been used for the analysis of mesenchymal stem cells (MSC). This study compared different protocols for fixing, storing, preserving, and establishing the correct numbers of dental derived MSC submitted to μ‐XRF analysis. Stem cells were obtained from human dental tissue. After cell expansion, and MACS isolation, the samples were fixed and the following quantities of cells 1 × 10⁴ to 1 × 10⁷ were divided in two groups: G1: fixed in 4% paraformaldehyde diluted in phosphate‐buffered saline solution, and G2: fixed in 4% paraformaldehyde diluted in MilliQ water. The G1 cells showed precipitation of chemical components from the solution resulting in the formation of salt crystals while G2 cells were clear and almost transparent in the sample holder. With regards to cells concentration, the best results occurred when four droplets of 1 × 10⁷ cells were analyzed. This work shows that to identify and study the distribution of trace elements in MSC by μ‐XRF, the best protocol is fixation in 4% paraformaldehyde diluted with MilliQ water at 4°C and a concentration of four incremental droplets of 1 × 10⁷ cells. Microsc. Res. Tech. 79:149–154, 2016. © 2016 Wiley Periodicals, Inc.     

Role of spermatogonial stem cells extract in transdifferentiation of 5‐Aza‐2′‐deoxycytidine‐treated bone marrow mesenchymal stem cells into germ‐like cells

As one of the induced pluripotent stem cells (iPSCs) methods, spermatogonial stem cells (SSC_S) extract is considered as new approach in stem cell therapy of infertility. 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) inhibits methyltransferase enzyme, and induces gene reprogramming; herein, the effects of SSC_S extract incubation in 5‐aza‐dC‐treated bone marrow mesenchymal stem cells (BMMSCs) has been surveyed. BMMSCs were isolated from femurs of three to four weeks old male NMRI mice, and the cells at passage three were treated with 2 µM 5‐aza‐dC for 72 hours. SSCs were isolated, cultured, and harvested at passage three to collect SSC_S extract; BMMSCs were then incubated with SSC_S extract in the three time periods: 72 hours, one week and two weeks. There were five groups: control, sham, extract, 5‐aza‐dC and extract‐5‐aza‐dC. After one week of incubation, flow cytometry and real‐time polymerase chain reaction (PCR) exhibited high levels of expression for β1‐ and α6‐integrins and promyelocytic leukaemia zinc finger (PLZF) in extract and extract‐5‐aza‐dC groups (P < 0.05 vs. control and 5‐aza‐dC), and cells in these two groups had two forms of morphology, round and fusiform, similar to germ‐like cells. 5‐aza‐dC had no significant effects during the three time periods of evaluation. These data disclose the effectiveness of SSCs extract incubation in transdifferentiation of BMMSCs into germ‐like cells; this strategy could introduce a new approach for treatment of male infertility in clinic. Microsc. Res. Tech. 79:365–373, 2016. © 2016 Wiley Periodicals, Inc.     

Stem cells in the development and differentiation of the human adrenal glands

There are no studies on stem cells (SCs) and development and differentiation (DD) of the human adrenal glands. The SCs in DD of the adrenal glands were herein investigated histochemically and immunohistochemically in 18 human embryonic adrenal glands at gestational week (GW) 7–40. At 7 GW, the adrenal glands were present, and at 7 GW, numerous embryonic SCs (ESCs) are seen to create the adrenal cortex. The ESCs were composed exclusively of small cells with hyperchromatic nuclei without nucleoli. The ESCs were positive for neural cell adhesion molecule, KIT, neuron‐specific enolase, platelet‐derived growth factor receptor‐α, synaptophysin, and MET. They were negative for other SC antigens, including chromogranin, ErbB2, and bcl‐2. They were also negative for lineage antigens, including cytokeratin (CK)7, CK8, CK18, and CK19, carcinoembryonic antigen, carbohydrate antigen 19‐9, epithelial membrane antigen, HepPar1, mucin core apoprotein (MUC)1, MUC2, MUC5AC, and MUC6, and cluster differentiation (CD)3, CD45, CD20, CD34, and CD31. The Ki‐67 labeling index (LI) was high (Ki‐67 LI = around 20%). α‐Fetoprotein was positive in the ESCs and adrenal cells. The ESC was first seen in the periphery of the adrenal cortex at 7–10 GW. The ESC migrates into the inner part of the adrenal cortex. Huge islands of ESC were present near the adrenal, and they appeared to provide the ESC of the adrenal. At 16 GW, adrenal medulla appeared, and the adrenal ESCs were present in the periphery or the cortex, in the cortical parenchyma, corticomedullary junctions, and in the medulla. The adrenal essential architecture was established around 20 GW; however, there were still ESCs. At term, there are a few ESCs. These data suggest that the adrenal glands were created by ESCs. Microsc. Res. Tech., 78:59–64, 2015. © 2014 Wiley Periodicals, Inc.     

Bones Morphogenic Protein‐4 and retinoic acid combined treatment comparative analysis for in vitro differentiation potential of murine mesenchymal stem cells derived from bone marrow and adipose tissue into germ cells

Nowadays, infertility is no longer considered as an unsolvable disorder due to progresses in germ cells derived from stem lineage with diverse origins. Technical and ethical challenges push researchers to investigate various tissue sources to approach more efficient gametes. The purpose of the current study is to investigate the efficacy of a combined medium, retinoic acid (RA) together with Bone Morphogenic Protein‐4 (BMP4), on differentiation of Bone Marrow Mesenchymal Stem Cells (BMMSCs) and adipose‐derived mesenchymal stem cells (ADMSCs) into germ cells. Murine MSCs were obtained from both Bone Marrow (BM) and Adipose Tissue (AT) samples and were analyzed for surface markers to get further verification of their nature. BMMSCs and ADMSCs were induced into osteogenic and adipogenic lineage cells respectively, to examine their multipotency. They were finally differentiated into germ cells using media enriched with BMP4 for 4 days followed by addition of RA for 7 days (11 days in total). Analyzing of differentiation potential of BMMSCs‐ and ADMSCs were performed via Immunofluorescence, Flowcytometry and Real time‐PCR techniques for germ cell‐specific markers (Mvh, Dazl, Stra8 and Scp3). Mesenchymal surface markers (CD90 and CD44) were expressed on both BMMSCs and ADMSCs, while endothelial and hematopoietic cell markers (CD31 and CD45) had no expression. Finally, all germ‐specific markers were expressed in both BM and AT. Although germ cells differentiated from ADMSCs showed faster growth and proliferation as well as easy collection, they significantly expressed germ‐specific markers lower than BMMSCs. This suggests stronger differentiation potential of murine BMMSCs than ADMSCs.     

The effects of human menstrual blood stem cells‐derived granulosa cells on ovarian follicle formation in a rat model of premature ovarian failure

Many studies have reported that human endometrial mesenchymal stem cells (HuMenSCs) are capable of repairing damaged tissues. The aim of the present study was to investigate the effects of HuMenSCs transplantation as a treatment modality in premature ovarian failure (POF) associated with chemotherapy‐induced ovarian damage. HuMenSCs were isolated from menstrual blood samples of five women. After the in vitro culture of HuMenSCs, purity of the cells was assessed by cytometry using CD44, CD90, CD34, and CD45 FITC conjugate antibody. Twenty‐four female Wistar rats were randomly divided into four groups: negative control, positive control, sham, and treatment groups. The rat models of POF used in our study were established by injecting busulfan intraperitoneally into the rats during the first estrus cycle. HuMenSCs were transplanted by injection via the tail vein into the POF‐induced rats. Four weeks after POF induction, ovaries were collected and the levels of Amh, Fst, and Fshr expression in the granulosa cell (GC) layer, as well as plasma estradiol (E2) and progesterone (P4) levels were evaluated. Moreover, migration and localization of DiI‐labeled HuMenSCs were detected, and the labeled cells were found to be localized in GCs layer of immature follicles. In addition to DiI‐labelled HuMenSCs tracking, increased levels of expression of Amh and Fshr and Fst, and the high plasma levels of E2 and P4 confirmed that HuMenSC transplantation had a significant effect on follicle formation and ovulation in the treatment group compared with the negative control (POF) group.     

Three‐dimensional Co‐culture of hepatic progenitor cells and mesenchymal stem cells in vitro and in vivo

Introduction: Here we co‐cultured hepatic progenitor cells (HPCs) and mesenchymal stem cells (MSCs) to investigate whether the co‐culture environments could increase hepatocytes form. Methods: Three‐dimensional (3D) co‐culture model of HPCs and MSCs was developed and morphological features of cells were continuously observed. Hepatocyte specific markers Pou5f1/Oct4, AFP, CK‐18 and Alb were analyzed to confirm the differentiation of HPCs. The mRNA expression of CK‐18 and Alb was analyzed by RT‐PCR to investigate the influence of co‐culture model to the terminal differentiation process of mature hepatocytes. The functional properties of hepatocyte‐like cells were detected by continuously monitoring the albumin secretion using Gaussia luciferase assays. Scaffolds with HPCs and MSCs were implanted into nude mouse subcutaneously to set up the in vivo co‐culture model. Results: Although two groups formed smooth spheroids and high expressed of CK‐18 and Alb, hybrid spheroids had more regular structures and higher cell density. CK‐18 and Alb mRNA were at a relatively higher expression level in co‐culture system during the whole cultivation time (P < 0.05). Albumin secretion rates in the hybrid spheroids had been consistently higher than that in the mono‐culture spheroids (P < 0.05). In vivo, the hepatocyte‐like cells were consistent with the morphological features of mature hepatocytes and more well‐differentiated hepatocyte‐like cells were observed in the co‐culture group. Conclusions: HPCs and MSCs co‐culture system is an efficient way to form well‐differentiated hepatocyte‐like cells, hence, may be helpful to the cell therapy of hepatic tissues and alleviate the problem of hepatocytes shortage. Microsc. Res. Tech. 78:688–696, 2015. © 2015 Wiley Periodicals, Inc.     

Ultrastructural study of mouse adipose‐derived stromal cells induced towards osteogenic direction

We investigated the ultrastructural characteristics of mouse adipose‐derived stem/stromal cells (ASCs) induced towards osteogenic lineage. ASCs were isolated from adipose tissue of FVB‐Cg‐Tg(GFPU)5Nagy/J mice and expanded in monolayer culture. Flow cytometry, histochemical staining, and electron microscopy techniques were used to characterize the ASCs with respect to their ability for osteogenic differentiation capacity. Immunophenotypically, ASCs were characterized by high expression of the CD44 and CD90 markers, while the relative content of cells expressing CD45, CD34 and CD117 markers was <2%. In assays of differentiation, the positive response to osteogenic differentiation factors was observed and characterized by deposition of calcium in the extracellular matrix and alkaline phosphatase production. Electron microscopy analysis revealed that undifferentiated ASCs had a rough endoplasmic reticulum with dilated cisterns and elongated mitochondria. At the end of the osteogenic differentiation, the ASCs transformed from their original fibroblast‐like appearance to having a polygonal osteoblast‐like morphology. Ultrastructurally, these cells were characterized by large euchromatic nucleus and numerous cytoplasm containing elongated mitochondria, a very prominent rough endoplasmic reticulum, Golgi apparatus and intermediate filament bundles. Extracellular matrix vesicles of variable size similar to the calcification nodules were observed among collagen fibrils. Our data provide the ultrastructural basis for further studies on the cellular mechanisms involved in osteogenic differentiation of mouse adipose‐derived stem/stromal cells. Microsc. Res. Tech. 79:557–564, 2016. © 2016 Wiley Periodicals, Inc.     

Spherical EBSD

Spheres, or more usually spherical surfaces, are important in electron backscatter diffraction. Both electron backscatter patterns (EBSPs) and pole figure texture data are more accurately represented on the spherical surface, S²; and unit quaternions, which are the optimal method for orientation calculations, exist on the surface of the hypersphere, S³. This paper is split into two distinct parts. The first shows a little of the history of the EBSP technique, including the use of spheres to assemble a spherical Kikuchi map (SKM) and as calibration artefacts. The second part relates new developments in the analysis of EBSPs on the surface of a sphere, a new spherical Hough transform and ideas for fully automatic, ab initio analysis of unknown phases using collections of EBSPs assembled as spherical Kikuchi maps.     

Leaf and stem micromorphology of Byrsonima sericea DC. by light and scanning electron microscopy

Micromorphological studies were carried out using multiple microscopic techniques on the leaves and stem bark of Byrsonima sericea DC. (Malpighiaceae), a species popularly known as “murici” and used medicinally, in order to identify both qualitative and quantitative features of leaf and stem anatomy and histochemistry as differential parameters to support both the quality control of its ethnodrugs and the taxonomy of the genus. The study was conducted using traditional techniques of plant anatomy, histochemical tests, and the stomatal index (SI). Byrsonima sericea has hypostomatic leaves, anomocytic stomata, and its epidermal walls are anticlinal and straight on the adaxial and curved on the abaxial faces. T‐shaped trichomes were observed mainly on the abaxial surface. The leaf epidermis showed waxes syntopism on both surfaces, with the occurrence of different crystalloid forms on a single phylloplane. The mesophyll is dorsiventral, with 3‐4 collateral vascular bundles. Phenolic compounds, starch, and proteins were identified in the petiole and stem. The SI was 14.5 ± 0.53% (p < .05), but did not showed significant variations. A set of characters were found to be distinctive for the studied species, however, constituting parameters that could be used to separate B. sericea from other species of the genus.     

Huge clusters of embryonic stem cells in human embryos: A morphologic study

Background: Nothing is known about huge clusters (HC) of embryonic stem cells (ESC) in human fetal organs (HFO). Aim: To know the status of HC‐ESC in HFO. Methods: Morphology and immunohistochemistry (IHC) in 32 HFO of 7–40 gestational weeks (GW). Results: HC‐ESC were seen in many HFO including central nervous system, spinal cords, spine, soft tissue, bone, skin, thyroid, lung, liver, pancreas, gall bladder, extrahepatic bile duct, adrenal, kidney, bladder, foregut, midgut, hindgut, female and male genital organs, and neurons. HC‐ESC's were composed of two populations depending on constituting cells. One were large cells with ample acidophilic cytoplasms with vesicular nuclei and nucleoli. The other were small cells with scant cytoplasm with hyperchromatic nuclei without nucleoli, resembling lymphocytes. The HC‐ESC were frequently showed neuronal differentiation. HC‐ESC were positive for NCAM, synaptophysin, NSE, chromogranin, PDGFRA, AFP, ErbB2, bcl‐2, KIT, MET. They were negative for CD45, CD3, CD20, EMA, CEA, CA19‐9, cytokeratin (CK) 7, CK8, CK18, CK19, MUC1, MUC2, MUC5AC, and MUC6. The mean Ki‐67 labeling index (LI) was 13% ± 7%. HC‐ESC showed a little glycogen but lacked mucins. These HC‐ESC were seen in 7–25 GW, and they were rarely seen in 26–40 GW. Conclusions: The morphology, IHC, and ontogeny of HC‐ESC were described. Microsc. Res. Tech. 77:825–831, 2014. © 2014 Wiley Periodicals, Inc.     

<i>miR-103-3p</i> regulates the differentiation of bone marrow mesenchymal stem cells in myelodysplastic syndrome

The pathogenesis of myelodysplastic syndrome (MDS) may be related to the abnormal expression of microRNAs (miRNAs), which could influence the differentiation capacity of mesenchymal stem cells (MSCs) towards adipogenic and osteogenic lineages. In this study, exosomes from bone marrow plasma were successfully extracted and identified. Assessment of miR-103-3p expression in exosomes isolated from BM in 34 MDS patients and 10 controls revealed its 0.52-fold downregulation in patients with MDS compared with controls (NOR) and was downregulated 0.55-fold in MDS-MSCs compared with NOR-MSCs. Transfection of MDS-MSCs with the miR-103-3p mimic improved osteogenic differentiation and decreased adipogenic differentiation in vitro, while inhibition of miR-103-3p showed the opposite results in NOR-MSCs. Thus, the expression of miR-103-3p decreases in MDS BM plasma and MDS-MSCs, significantly impacting MDS-MSCs differentiation. The miR-103-3p mimics may boost MDS-MSCs osteogenic differentiation while weakening lipid differentiation, thereby providing possible target for the treatment of MDS pathogenesis.     

Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies

Stereology applied on histological sections is the ‘gold standard’ for obtaining quantitative information on cancellous bone structure. Recent advances in micro computed tomography (µCT) have made it possible to acquire three-dimensional (3D) data non-destructively. However, before the 3D methods can be used as a substitute for the current ‘gold standard’ they have to be verified against the existing standard. The aim of this study was to compare bone structural measures obtained from 3D µCT data sets with those obtained by stereology performed on conventional histological sections using human tibial bone biopsies. Furthermore, this study forms the first step in introducing the proximal tibia as a potential bone examination location by peripheral quantitative CT and CT. Twenty-nine trabecular bone biopsies were obtained from autopsy material at the medial side of the proximal tibial metaphysis. The biopsies were embedded in methylmetacrylate before µCT scanning in a Scanco µCT 40 scanner at a resolution of 20 × 20 × 20 µm³, and the 3D data sets were analysed with a computer program. After µCT scanning, 16 sections were cut from the central 2 mm of each biopsy and analysed with a computerized method. Trabecular bone volume (BV/TV) and connectivity density (CD) were estimated in both modalities, whereas trabecular bone pattern factor (TBPf) was estimated on the histological sections only. Trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), and structure model index (SMI) were estimated with the µCT method only. Excellent correlations were found between the two techniques for BV/TV (r = 0.95) and CD (r = 0.95). Additionally, an excellent relationship (r = 0.95) was ascertained between TBPf and SMI. The study revealed high correlations between measures of bone structure obtained from conventional 2D sections and 3D µCT data. This indicates that 3D µCT data sets can be used as a substitute for conventional histological sections for bone structural evaluations.