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.     

Effects of human umbilical cord mesenchymal stem cells on co‐cultured ovarian carcinoma cells

Ovarian carcinoma is mainly treated by surgery aided by chemotherapy. If supplemented by stem cells treatment, its recurrence rate and mortality rate will be decreased. This is a new therapy. In this study, ovarian cancer cells were cultured together with umbilical cord mesenchymal stem cells (UCMSCs), and the interactions between them were observed. The results showed that the survival rates of UCMSCs increased to 83.8 ± 2.2% from 56.5 ± 5.5%, and the survival rates of ovarian cancer cells decreased to 16.2 ± 2.2% from 43.5 ± 5.5% with the progression of the cultural time from 24 to 96 hr. There was a significant difference between them (p < .05). It revealed that UCMSCs could inhibit the proliferation of ovarian cancer cells.     

New insights into the cellular makeup and progenitor potential of palatal connective tissues

The present study investigated the regenerative potential of connective tissues harvested from two palatal areas widely used as donor sites for muco‐gingival surgical approaches. Connective tissue grafts (CTGs) were obtained by de‐epithelialisation of a free gingival graft (deCTG) and by a split flap approach from a previous donor site (reCTG). Two types of mesenchymal stem cell (MSCs) were isolated and were named de‐epithelialised MSCs (deMSCs) and re‐entry MSCs (reMSCs). The cells were characterised and cellular functionality was investigated. CTGs were evaluated using immunohistochemical and ultrastructural approaches. No significant differences were observed regarding the frequency of colony‐forming unit‐ fibroblasts, migration potential, and population doubling time between the two cell lines (p > 0.05). Both cell lines showed positivity for CD105, CD73, CD90, and CD44 and negative expression for CD34/45, CD14, CD79a, and HLA‐DR. MSCs from both cell lines successfully differentiated into osteogenic, adipogenic, and chondrogenic lineages. Cells expressing antigens characteristic of CD34+ stromal cells (CD34+, αSMA?, CD31?) were traced in both CTGs. Ultrastructural analysis highlighted the presence of putative progenitors, namely fibroblasts,—in the pericapillary regions and in remote regions of the lamina propria‐ and pericytes—surrounding the capillaries. This study provides supplementary arguments for the use of CTG grafts in clinical practice due to the presence of putative progenitor cell. However, results were inconclusive regarding clinical decision‐making to determine optimal harvesting area. Prior harvesting in the donor area did not appear to alter the regenerative capabilities of the connective tissue.     

Morphological and morphometric characterization of agoutis' peripheral blood cells (Dasyprocta prymnolopha, Wagler, 1831) raised in captivity

Thirty adult agoutis (Dasyprocta primnolopha) from the Nucleus of Study and Preservation of Wild Animals at the Federal University of Piauí were used. Blood scrubs of these animals were colored by the Leishman method and analyzed in light microscopy. The cells had been measured using programs that analyze images (Leica QWin - Image Processing and Analysis Software). Mature erythrocytes, basophil reticulocytes, lymphocytes, eosinophils, neutrophils, monocytes, and thrombocytes were identified. Agoutis' erythrocytes presented elliptical form, without nucleus with an average diameter of 5.64 micromeres ± 0.38. The lymphocytes are spherical cells with scarce cytoplasm, dense and with a very centralized rounded nucleus measuring an average diameter of 13.20 micromeres ± 0.35. The monocytes are slightly basophilic, with a spherical nucleus, central constriction, and an average diameter of 20.59 micromeres ± 0.32. The neutrophils are spherical, with a polymorphic lobulated nucleus, with an average diameter of 11.2 micromeres ± 0.20. The eosinophils are spherical with lobulated nucleus and with an average diameter of 14.25 micromeres ± 0.36. Only five basophils were observed, with abundance of cytoplasmic granules with 9.8 micrometers of diameter ± 0.30. Thrombocytopenic pleomorphism was frequent. There were similarities in the cellular constituents in peripheral blood of agoutis and of other rodents and humans. The cellular types from the peripheral blood, the morphology, and morphometry of the blood's cells did not vary according to sex.     

Human umbilical cord blood mesenchymal stem cells conditioned media inhibits hypoxia-induced apoptosis in H9c2 cells by activation of the survival protein Akt

This work aimed to study the beneficial role of human umbilical cord blood-derived mesenchymal stem cellconditioned medium (MSC-CM) in hypoxia-induced apoptosis in H9c2 cardiomyoblasts, in which the serine/heroinekinases (Akt) pathway would be involved. For this, CM was collected by culturing MSCs in serum-free DMEMmedium for 24 h, and paracrine factors were analyzed by protein chip. H9c2 cells were divided into the followinggroups: control group, hypoxia group, MSC-CM intervention group (CM group), MSC-CM + Akt phosphorylationinhibitor (LY294002) group (LY group). Apoptosis of the H9c2 cells was tested with chromatin dye Hoechst 33342and FITC-conjugated Annexin V apoptosis detection kit by flow cytometer after a hypoxia/serum deprivation (H/SD)for 24 h. The apoptosis-related proteins were evaluated by Western blot. MSC-CM displayed significantly elevatedlevels of growth factors, anti-inflammatory, and anti-apoptosis cytokines. On Hoechst 33342 apoptosis staining, theH9c2 cell morphology displayed a lower proportion of apoptosis in the CM group than those in the hypoxia group,while apoptosis was increased in LY group. Flow cytometer analysis revealed the apoptosis ratio in the CM group waslower than the hypoxia group (12.34 ± 2.00% vs. 21.73 ± 2.58%; p < 0.05), while the LY group was significantly higher(22.54 ± 3.89%). Active caspase-3 expression was increased in hypoxia group than control group (p < 0.05), butdecreased in CM group (p < 0.01). Umbilical cord blood-derived mesenchymal stem cell-conditioned media secretemultiple paracrine factors that are able to inhibit hypoxia-induced H9c2 cardiomyoblasts apoptosis, and in which theactivation of Akt phosphorylation is involved to achieve the protective effect.     

Nanoscale imaging of morphological changes of umbilical cord in pre-eclampsia

It is known that pre‐eclampsia affects the structure of the umbilical cord including changes in diameter and wall thickness. In this work, the morphological changes of umbilical cords associated with pre‐eclampsia were investigated using scanning electron microscopy (SEM) and atomic force microscopy (AFM). The SEM images showed the overall structural changes in the umbilical cord, and the AFM imaged the surface of the cord in the nanometer range. The amount of Wharton's jelly was reduced in the cords of pre‐eclampsia patients and it was holed along the boundary. Compared to a normal pregnancy, the surface of a pre‐eclampsia cord was relatively smooth. In all components (Wharton's jelly, veins, and arteries), the values for surface roughness, Sa (average value of the roughness), Sq (root mean square), and Sz (peak to peak value), were smaller than those of the control (P < 0.05). Especially, the values for Sa of veins were ～fourfold less than those of the controls (P < 0.05). In pre‐eclamptic cords, the amount of elastin in veins was increased while that of the artery was decreased. Microsc. Res. Tech. 2012. © 2012 Wiley Periodicals, Inc.     

Ultrastructural analysis of murine hippocampal neural progenitor cells in culture

Despite the great number of studies devoted to neural stem/progenitor cell biology, the ultrastructural characteristics of these cells in vitro have not been fully studied. To determine the fine structure of hippocampal neural progenitor cells (NPCs) in culture, mouse fetal hippocampi (E18) were extracted, dissected, and cells were expanded as adherent monolayer culture. Electron microscopy revealed that NPCs had an immature phenotype, with a high nuclear/cytoplasmic ratio, small and scant organelles, underdeveloped endoplasmic reticulum, and many free ribosomes and polysomes. Our results may contribute to a better understanding of the fine structure and physiology of hippocampal NPCs in vitro. Microsc. Res. Tech. 78:128–133, 2015. © 2014 Wiley Periodicals, Inc.     

Comparative characterization of human fetal neural stem cells and induced neural stem cells from peripheral blood mononuclear cells

Human-induced neural stem cells (iNSCs) transplantation is a potential treatment of neurodegenerationdiseases. However, whether the reprogrammed cells have the same characterizations as human fetal neural stem cellsneeds further exploration. Here we isolated human fetal neural stem cells from aborted 12-week fetal brains andcompared with iNSCs reprogrammed from human peripheral blood mononuclear cells in gene expression, proliferationability, differentiation capacity, and the responses to tumor necrosis factor-α. We found that iNSCs and NSCs bothexpressed neural stem cell markers Nestin, SOX1, and SOX2. However, only iNSCs can be patterned into dopaminergicneurons and motor neurons. Furthermore, both iNSCs and NSCs can differentiate into oligodendrocyte progenitorcells. In addition, a low dose of tumor necrosis factor-α did not inhibit the proliferation and differentiation of iNSCsand NSCs. In conclusion, iNSCs have properties similar to, and even better than, fetal neural stem cells and may besuitable for disease modeling and transplantation.     

Ultrastructural characterization of apoptotic granulosa cells in caprine ovary

The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.     

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.     

Morphological and ultrastructural characterization of ionoregulatory cells in the teleost oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique

Aspects of ionoregulatory or mitochondria‐rich cell (MRC) differentiation and adaptation in Nile tilapia yolk‐sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole‐mount Nile tilapia larvae using anti‐ Na⁺/K⁺‐ATPase as a primary antibody and Fluoronanogold? (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk‐sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺‐ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level. Microsc. Res. Tech., 76:1016–1024, 2013. © 2013 Wiley Periodicals, Inc.     

Multifractal characterization of morphology of human red blood cells membrane skeleton

The purpose of this paper is to show applicability of multifractal analysis in investigations of the morphological changes of ultra‐structures of red blood cells (RBCs) membrane skeleton measured using atomic force microscopy (AFM). Human RBCs obtained from healthy and hypertensive donors as well as healthy erythrocytes irradiated with neutrons (45 μGy) were studied. The membrane skeleton of the cells was imaged using AFM in a contact mode. Morphological characterization of the three‐dimensional RBC surfaces was realized by a multifractal method. The nanometre scale study of human RBCs surface morphology revealed a multifractal geometry. The generalized dimensions D_q and the singularity spectrum f(α) provided quantitative values that characterize the local scale properties of their membrane skeleton organization. Surface characterization was made using areal ISO 25178‐2: 2012 topography parameters in combination with AFM topography measurement. The surface structure of human RBCs is complex with hierarchical substructures resulting from the organization of the erythrocyte membrane skeleton. The analysed AFM images confirm a multifractal nature of the surface that could be useful in histology to quantify human RBC architectural changes associated with different disease states. In case of very precise measurements when the red cell surface is not wrinkled even very fine differences can be uncovered as was shown for the erythrocytes treated with a very low dose of ionizing radiation.     

Growth factors, CD34 positive cells, and fibrin network analysis in concentrated growth factors fraction

An interesting clinical option for optimizing healing tissue is the use of platelet concentrate. Platelets contain high quantities of growth factors, among these TGF-β1 and VEGF, which are known to be implicated in tissue regeneration. CGF is produced by processing blood samples with a special centrifuge device; three layers are formed: top acellular plasma (PPP), middle CGF and bottom red blood cells (RBC) layers. Given that to date there are no data concerning the biological characteristic of CGF, the aim of this study was to evaluate the presence of TGF-β1 and VEGF in CGF and also in PPP and RBC layers. In addition, since circulating stem cells are recruited from blood to injured tissue for healing we also evaluated the presence of CD34 positive cells. Our data show the presence of TGF-β1 and VEGF in CGF and RBC layers. In addition, we show CD34 positive cells in CGF.     

两种不同淋巴细胞分离液对脐带血单核细胞的提取及后期诱导培养CIK细胞的影响

目的：研究两种不同的淋巴细胞分离液对脐带血单核细胞的分离效果及后期对CIK细胞的诱导培养的影响。方法：分别使用国产TBD以及进口GE healthcare牌淋巴细胞分离液分离提取脐带血单核细胞,用悬浮细胞培养法诱导培养脐带血单核细胞形成CIK细胞,再用血球计数板及台盼兰染色法检测细胞密度及活率。结果：国产的TBD牌所分离的单核细胞分层较清楚,并且方便提取,而使用进口GE healthcare的淋巴细胞分离液提取的单核细胞数量较多,但经过后期诱导培养CIK细胞发现两种分离液提取的细胞培养到后期数量逐渐接近。结论：国产TBD牌淋巴细胞分离液在用于脐带血淋巴细胞分离提取时较进口GE healthcare牌经济,并且细胞提取方便,后期培养效果无明显差别。     

Transplantation of BMP-7 gene-transfected bone marrow mesenchymal stem cells for the treatment of spinal cord injury in rats

Background: Spinal cord injury (SCI) is a serious traumatic disease of the central nervous system, and there is currently no effective treatment for SCI because of its complicated pathophysiology. Bone marrow mesenchymal stem cells (BMSCs) have multidirectional differentiation abilities. Our study aims to explore the effects of bone morphogenetic protein 7 (BMP-7)-modified BMSCs transplantation on the repair of SCI in rats. Methods: In this study, a rat spinal cord injury model was established with the modified Allen method. Then, BMSCs transfected with the BMP7 gene were transplanted to treat the spinal cord injury in rats. Forty Sprague-Dawley rats were randomly divided into the sham operation group (sham group), spinal cord injury group (model group), BMSC treatment group (BMSC group) and LV-BMP7-BMSC treatment group (LV-BMP7-BMSC group). The Basso, Beattie, and Bresnahan (BBB) score was used to evaluate the recovery of hindlimb function in the rats. The levels of neurofilament protein NF-200 (NF-200) and glial fibrillary acidic protein (GFAP) were detected by immunofluorescence, RT-PCR and Western blotting. Results: At 14 d, 21 d, and 28 d after treatment, the BBB score of the rats in the LV-BMP7-BMSC group was higher than that of the rats in the model group and BMSC group. The results showed that NF-200 was expressed at the local spinal cord injury site. Compared with that of the sham group, the NF-200 expression level of the BMSC group and LV-BMP7-BMSC group was increased (P < 0.05). The results showed that the mRNA expression levels of NF-200 in the spinal cord tissue of the BMSC group and LV-BMP7-BMSC group were increased compared with those of the sham group (P < 0.05). The western blotting results further confirmed the PCR results. Conclusion: BMP-7 gene-modified BMSC transplantation can promote the repair of spinal cord functions after SCI in rats.     

Differentiation of Leydig cells in the Mongolian gerbil

Information on postnatal Leydig cell (LC) differentiation in the Mongolian gerbil has been unavailable. Therefore, current investigation was designed to examine LC lineage differentiationin this rodent, from birth to adulthood. Gerbil testes at 1 day, 1–7 weeks (w), 2 and 3 months of age were conventionally processed by light and transmission electron microscopy. Immunocytochemistry for specific markers of steroidogenic enzymes, namely 3β‐hydroxysteroid dehydrogenase (3β‐HSD) and 11β‐hydroxysteroid steroid dehydrogenase 1 (11β‐HSD1) and also for androgen receptor (AR) was performed. The establishment of adult Leydig cell populations (ALC) during testis maturation in the gerbil follows the pattern previously described in other mammalian species, with the four progressive stages of differentiation. The LC progenitors were identified at second w by 3β‐HSD expression; the first newly formed ALC were recognized at fourth w whereas immature ALC appeared at fifth w. The latter were recognized by abundance of cytoplasmic lipid, in addition to expression of 11β‐HSD1 and intense nuclear AR immunoreaction. Mature ALC in gerbil exhibited irregular eccentric nuclei and a cytoplasmic canaliculus in the perinuclear area. Only one third of mature ALC in adult gerbils showed a high expression of 11β‐HSD1 and AR expression was highly variable among them. In conclusion, the process of differentiation of ALC population in gerbil follows the pattern previously established for other rodents. However, the resulting mature ALC are strikingly different due their singular asymmetric morphology and presence of a cytoplasmic canaliculus and as well as their functional heterogeneity. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Measuring the velocity of fluorescently labelled red blood cells with a keyhole tracking algorithm

In this paper we propose a tracking algorithm to measure the velocity of fluorescently labelled red blood cells travelling through microvessels of tumours, growing in dorsal skin flap window chambers, implanted on mice. Preprocessing removed noise and artefacts from the images and then segmented cells from background. The tracking algorithm is based on a ‘keyhole’ model that describes the probable movement of a segmented cell between contiguous frames of a video sequence. When a history of cell movement exists, past, present and a predicted landing position of the cells will define two regions of probability that resemble the shape of a keyhole. This keyhole model was used to determine if cells in contiguous frames should be linked to form tracks and also as a postprocessing tool to join split tracks and discard links that could have been formed due to noise or uncertainty. When there was no history, a circular region around the centroid of the parent cell was used as a region of probability. Outliers were removed based on the distribution of the average velocities of the tracks. Since the position and time of each cell is recorded, a wealth of statistical measures can be obtained from the tracks. The algorithm was tested on two sets of experiments. First, the vasculatures of eight tumours with different geometries were analyzed; average velocities ranged from 86 to 372 μm s^?1, with minimum and maximum track velocities 7 and 1212 μm s^?1, respectively. Second, a longitudinal study of velocities was performed after administering a vascular disrupting agent to two tumours and the time behaviour was analyzed over 24 h. In one of the tumours there is a complete shutdown of the vasculature whereas in the other there is a clear decrease of velocity at 30 min, with subsequent recovery by 6 h. The tracking algorithm enabled the simultaneous measurement of red blood cell velocity in multiple vessels within an intravital video sequence, enabling analysis of heterogeneity of flow and response to treatment in mouse models of cancer.     

Automatic segmentation,counting, size determination and classification of white blood cells

The counts, the so-called differential counts, and sizes of different types of white blood cells provide invaluable information to evaluate a wide range of important hematic pathologies from infections to leukemia. Today, the diagnosis of diseases can still be achieved mainly by manual techniques. However, this traditional method is very tedious and time-consuming. The accuracy of it depends on the operator’s expertise. There are laser based cytometers used in laboratories. These advanced devices are costly and requires accurate hardware calibration. They also use actual blood samples. Thus there is always a need for a cost effective and robust automated system. The proposed system in this paper automatically counts the white blood cells, determine their sizes accurately and classifies them into five types such as basophil, lymphocyte, neutrophil, monocyte and eosinophil. The aim of the system is to help for diagnosing diseases. In our work, a new and completely automatic counting, segmentation and classification process is developed. The outputs of the system are the number of white blood cells, their sizes and types.