Search results for: stem cell migration

Authors: Hirowati Ali, Aisyah Ellyanti, Dewi Rusnita, Septelia Inawati Wanandi

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Stem cell has been known for its potency to be differentiated in many cells. Recently stem cell has been used for many treatment of degenerative medicine. It is still controversy whether stem cell can be used for therapy or these cells can activate cancer stem cell. SCUBE2 is a novel secreted and membrane-anchored protein which has been reported to its role in better prognosis and inhibition of cancer cell proliferation. Our study aims to observe whether stem cell can up-regulate SCUBE2 gene in MCF7 breast cancer cell line. We used in vitro study using MCF-7 cell treated with stem cell derived from placenta Wharton's jelly which has been known for its stemness and widely used. Our results showed that MCF-7 cell line grows up rapidly in 6-well culture dish. Stem cell was cultured in 6-well dish. After 50%-60% MCF-7 confluence, we co-cultured these cells with stem cells for 24 hours and 48 hours. We hypothesize SCUBE2 gene which is previously known for its higher expression in better prognosis of breast cancer, is up-regulated after stem cells addition in MCF7 culture dishes.

Keywords: breast cancer cells, inhibition of cancer cells, mesenchymal stem cells, SCUBE2

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Authors: Nishanth Venugopal Menon, Chuah Yon Jin, Samantha Phey, Wu Yingnan, Zhang Ying, Vincent Chan, Kang Yuejun

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Cell Migration is a vital phenomenon that the cells undergo in various physiological processes like wound healing, disease progression, embryogenesis, etc. Cell migration depends primarily on the chemical and physical cues available in the cellular environment. The chemical cue involves the chemokines secreted and gradients generated in the environment while physical cues indicate the impact of matrix properties like nanotopography and stiffness on the cells. Mesenchymal Stem Cells (MSCs) have been shown to have a role wound healing in vivo and its migration to the site of the wound has been shown to have a therapeutic effect. In the field of stem cell based tissue regeneration of bones and cartilage, one approach has been to introduce scaffold laden with MSCs into the site of injury to enable tissue regeneration. In this work, we have studied the combinatorial impact of the substrate physical properties on MSC migration. A microfluidic in vitro model was created to perform the migration studies. The microfluidic model used is a three compartment device consisting of two cell seeding compartments and one migration compartment. Four different PDMS substrates with varying substrate roughness, stiffness and hydrophobicity were created. Its surface roughness and stiffness was measured using Atomic Force Microscopy (AFM) while its hydrphobicity was measured from the water contact angle using an optical tensiometer. These PDMS substrates are sealed to the microfluidic chip following which the MSCs are seeded and the cell migration is studied over the period of a week. Cell migration was quantified using fluorescence imaging of the cytoskeleton (F-actin) to find out the area covered by the cells inside the migration compartment. The impact of adhesion proteins on cell migration was also quantified using a real-time polymerase chain reaction (qRT PCR). These results suggested that the optimal substrate for cell migration would be one with an intermediate level of roughness, stiffness and hydrophobicity. A higher or lower value of these properties affected cell migration negatively. These observations have helped us in understanding that different substrate properties need to be considered in tandem, especially while designing scaffolds for tissue regeneration as cell migration is normally impacted by the combinatorial impact of the matrix. These observations may lead us to scaffold optimization in future tissue regeneration applications.

Keywords: cell migration, microfluidics, in vitro model, stem cell migration, scaffold, substrate properties

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Authors: Vytautas Galvanauskas, Vykantas Grincas, Rimvydas Simutis

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This paper investigates a possible practical solution for reasonable oxygen supply during the pluripotent stem cells expansion processes, where the stem cells propagate as aggregates in stirred-suspension bioreactors. Low glucose and low oxygen concentrations are preferred for efficient proliferation of pluripotent stem cells. However, strong oxygen limitation, especially inside of cell aggregates, can lead to cell starvation and death. In this research, the oxygen concentration profile inside of stem cell aggregates in a stem cell expansion process was predicted using a modified oxygen diffusion model. This profile can be realized during the stem cells cultivation process by manipulating the oxygen concentration in inlet gas or inlet gas flow. The proposed approach is relatively simple and may be attractive for installation in a real pluripotent stem cell expansion processes.

Keywords: aggregated stem cells, dissolved oxygen profiles, modeling, stirred-tank, 3D expansion

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Authors: Hyo-Min Kim, Seokjin Ham, Mi-Joung Yoo, Minseon Kim, Tae-Young Roh

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The gastrointestinal (GI) tract of most animals, including murine, is highly compartmentalized epithelia which also provide distinct different functions of its own tissue. Nevertheless, these epithelia share certain characteristics that enhance immune responses to infections and maintain the barrier function of the intestine. GI tract epithelia also undergo regeneration not only in homeostatic conditions but also in a response to the damage. A full turnover of the murine gastrointestinal epithelium occurs every 4-5 day, a process that is regulated and maintained by a minor population of Lgr5+ adult stem cell that commonly conserved in the bottom of crypts through GI tract. Maintenance of the stem cell is somehow regulated by epigenetic factors according to recent studies. Chromatin vacancy, remodelers, histone variants and histone modifiers could affect adult stem cell fate. In this study, Lgr5-EGFP reporter mouse was used to take advantage of exploring the epigenetic dynamics among Lgr5 positive mutual stem cell in GI tract. Cells were isolated by fluorescence-activated cell sorting (FACS), gene expression levels, chromatin accessibility changes and histone modifications were analyzed. Some notable chromatin structural related epigenetic variants were detected. To identify the overall cell-cell interaction inside the stem cell niche, an extensive genome-wide analysis should be also followed. According to the results, nevertheless, we expected a broader understanding of cellular niche maintaining stem cells and epigenetic barriers through conserved stem cell in GI tract. We expect that our study could provide more evidence of adult stem cell plasticity and more chances to understand each stem cell that takes parts in certain organs.

Keywords: adult stem cell, epigenetics, LGR5 stem cell, gastrointestinal tract

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Authors: Jung Park, Anca Mazare, Klaus Von Der Mark, Patrik Schmuki

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In clinical application of TiO2 implants on tooth and hip replacement, migration, adhesion and differentiation of neighboring mesenchymal stem cells onto implant surfaces are critical steps for successful bone regeneration. In a recent decade, accumulated attention has been paid on nanoscale electrochemical surface modifications on TiO2 layer for improving bone-TiO2 surface integration. We generated, on titanium surfaces, self-assembled layers of vertically oriented TiO2 nanotubes with defined diameters between 15 and 100 nm and here we show that mesenchymal stem cells finely sense TiO2 nanotubular geometry and quickly decide their cell fate either to differentiation into osteoblasts or to programmed cell death (apoptosis) on TiO2 nanotube layers. These cell fate decisions are critically dependent on nanotube size differences (15-100nm in diameters) of TiO2 nanotubes sensing by integrin clustering. We further demonstrate that nanoscale topography-sensing is feasible not only in mesenchymal stem cells but rather seems as generalized nanoscale microenvironment-cell interaction mechanism in several cell types composing bone tissue network including osteoblasts, osteoclast, endothelial cells and hematopoietic stem cells. Additionally we discuss the synergistic effect of simultaneous stimulation by nanotube-bound growth factor and nanoscale topographic cues on enhanced bone regeneration.

Keywords: TiO2 nanotube, stem cell fate decision, nano-scale microenvironment, bone regeneration

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Authors: Nathalie Baeza-Kallee, Raphaël Bergès, Victoria Hein, Stéphanie Cabaret, Jeremy Garcia, Abigaëlle Gros, Emeline Tabouret, Aurélie Tchoghandjian, Carole Colin, Dominique Figarella-Branger

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Glioblastoma (GBM) contains cancer stem cells that are resistant to treatment. GBM cancer stem cell expresses glycolipids recognized by the A2B5 antibody. A2B5, induced by the enzyme ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyl transferase 3 (ST8Sia3), plays a crucial role in the proliferation, migration, clonogenicity, and tumorigenesis of GBM cancer stem cells. Our aim was to characterize the resulting effects of neuraminidase that remove A2B5 in order to target GBM cancer stem cells. To this end, we set up a GBM organotypic slice model; quantified A2B5 expression by flow cytometry in U87-MG, U87-ST8Sia3, and GBM cancer stem cell lines, treated or not by neuraminidase; performed RNAseq and DNA methylation profiling; and analyzed the ganglioside expression by liquid chromatography-mass spectrometry in these cell lines, treated or not with neuraminidase. Results demonstrated that neuraminidase decreased A2B5 expression, tumor size, and regrowth after surgical removal in the organotypic slice model but did not induce a distinct transcriptomic or epigenetic signature in GBM CSC lines. RNAseq analysis revealed that OLIG2, CHI3L1, TIMP3, TNFAIP2, and TNFAIP6 transcripts were significantly overexpressed in U87-ST8Sia3 compared to U87-MG. RT-qPCR confirmed these results and demonstrated that neuraminidase decreased gene expression in GBM cancer stem cell lines. Moreover, neuraminidase drastically reduced ganglioside expression in GBM cancer stem cell lines. Neuraminidase, by its pleiotropic action, is an attractive local treatment against GBM.

Keywords: cancer stem cell, ganglioside, glioblastoma, targeted treatment

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Authors: Celso Afonso Klein Jr., Henrique Cantarelli, Fernando Portella, Keiichi Hosaka, Eduardo Reston, Fabricio Collares, Roberto Zimmer

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TTo evaluate the influence of preheating self-adhesive cement at 39ºC on cell migration, cytotoxicity and degree of conversion. RelyX U200, Set PP and MaxCem Elite were subjected to a degree of conversion analysis (FTIR-ATR). For the cytotoxicity analysis, extracts (24 h and 7 days) were placed in contact with NIH/3T3 cells. For cell migration, images were captured of each sample until the possible closure of the cleft occurred. In the results of the degree of conversion, preheating did not improve the conversion of cement. For the MTT, preheating did not improve the results within 24 hours. However, it generated positive results within 7 days for the Set PP resin cement. For cell migration, high rates of cell death were found in all groups. It is concluded that preheating at 39ºC caused a positive effect only in increasing the cell viability of the Set PP resin cement and that both materials analyzed are highly cytotoxic.

Keywords: dental cements, resin cements, degree of conversion, cytotoxicity, cell migration assays

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Authors: S. AliReza Mirghasemi, Zameer Hussain, Mohammad Saleh Sadeghi, Narges Rahimi Gabaran, Mohamadreza Baghaban Eslaminejad

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Background: Despite promising results have shown by osteogenic cell-based demineralized bone matrix composites, they need to be optimized for grafts that act as structural frameworks in load-bearing defects. The purpose of this experiment is to determine the effect of bone-marrow-mesenchymal-stem-cells seeding on partially demineralized laser-perforated structural allografts that have been implanted in critical femoral defects. Materials and Methods: P3 stem cells were used for graft seeding. Laser perforation in four rows of three holes was achieved. Cell-seeded grafts were incubated for one hour until they were planted into the defect. We used four types of grafts: partially demineralized only (Donly), partially demineralized stem cell seeded (DST), partially demineralized laser-perforated (DLP), and partially demineralized laser-perforated stem cell seeded (DLPST). histologic and histomorphometric analysis were performed at 12 weeks. Results: Partially demineralized laser-perforated had the highest woven bone formation within graft limits, stem cell seeded demineralized laser-perforated remained intact, and the difference between partially demineralized only and partially demineralized stem cell seeded was insignificant. At interface, partially demineralized laser-perforated and partially demineralized only had comparable osteogenesis, but partially demineralized stem cell seeded was inferior. The interface in stem cell seeded demineralized laser-perforated was almost replaced by distinct endochondral osteogenesis with higher angiogenesis in the vicinity. Partially demineralized stem cell seeded and stem cell seeded demineralized laser-perforated graft surfaces had extra vessel-ingrowth-like porosities, a sign of delayed resorption. Conclusion: This demonstrates that simple cell-based composites are not optimal and necessitates the supplementation of synergistic stipulations and surface changes.

Keywords: structural bone allograft, partial demineralization, laser perforation, mesenchymal stem cell

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Authors: Ana M. Lara, Manuela Llano, Felipe Gaitán, Rosa H. Bustos, Ana Maria Perdomo-Arciniegas, Ximena Bonilla

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Umbilical cord blood is used as a source of progenitor and stem cells for the regeneration of the hematopoietic and immune system to treat patients with different hematological or non-hematological diseases. This stem cell source represents an advantage over the use of bone marrow or mobilized peripheral blood because it has a lower incidence rate of graft-versus-host disease, probably due to fewer immunological compatibility restrictions. However, its low cellular dose limits its use in pediatric patients. This work proposes the standardization of a cell expansion technique to compensate for the dose of infused cells through the ex-vivo manipulation of hematopoietic progenitor cells from umbilical cord blood before transplantation. The expansion model is carried out through co-cultures with mesenchymal stem cells (MSC) from bone marrow (BM) and less explored fetal tissues such as Wharton's jelly (WJ) and umbilical cord blood (UCB). Initially, a master cell bank of primary mesenchymal stem cells isolated from different sources was established and characterized following International Society of Cell Therapies (ISCT) indications. Additionally, we assessed the effect of a short 25 Gy cycle of gamma irradiation on cell cycle arrest of mesenchymal cells over the support capacity for the expansion of hematopoietic stem cells from umbilical cord blood was evaluated. The results show that co-cultures with MSC from WJ and UCB allow the cellular dose of HSPC to be maximized between 5 and 16 times having a similar support capacity as BM. In addition, was evaluated the hematopoietic stem progenitor cell's HSPC functionality through the evaluation of migration capacity, their differentiation capacity during culture time by flow cytometry to evaluate the expression of membrane markers associated with lineage-committed progenitors, their clonogenic potential, and the evaluation of secretome profile in the expansion process was evaluated. So far, the treatment with gamma irradiation maintains the hematopoietic support capacity of mesenchymal stem cells from the three sources studied compared to treatments without irradiation, favoring the use of fetal tissues that are generally waste to obtain mesenchymal cell lines for ex-vivo expansion systems. With the results obtained, a standardized protocol that will contribute to the development of ex-vivo expansion with MSC on a larger scale will be achieved, enabling its clinical use and expanding its application in adults.

Keywords: ex-vivo expansion, hematopoietic stem cells, hematopoietic stem cell transplantation, mesenchymal stem cells, umbilical cord blood

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Authors: Mojdeh Mohseni

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In the context of tissue engineering, the effect of microtopography as afforded by scaffold morphology is an important design parameter. Since the morphology of pores can effect on cell behavior, in this study, porous Chitosan (CHIT) - Gelatin (GEL)- Alginate (ALG) scaffolds with microtubule orientation structure were manufactured by unidirectional freeze-drying method and the effect of pore morphology on differentiation of Mesenchymal Stem Cells (MSCs) was investigated. This study showed that, the provided scaffold with natural polymer had good properties for cell behavior and the pores with highest orientation rate have produced appropriate substrate for the differentiation of stem cells.

Keywords: Chitosan, gelatin, Alginate, pore morphology, stem cell differentiation

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Authors: Ahmed Khalaf Reyad Raslan

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Cancer stem cells (CSCs) describe a class of pluripotent cancer cells that behave analogously to normal stem cells in their ability to differentiate into the spectrum of cell types observed in tumors. The de-differentiation processes, such as an epithelial-mesenchymal transition (EMT), are known to enhance cellular plasticity. Here, we demonstrate a new hypothesis to use hybrid superparamagnetic magnetite nanoparticles (Fe₃O₄)- graphene oxide functionalized surface with Collagen to target the cancer stem cell as an early detection tool for cancer. We think that with the use of magnetic resonance imaging (MRI) and the new hybrid system would be possible to track the cancer stem cells.

Keywords: hydrogel, alginate, reduced graphene oxide, collagen

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Authors: Ahmad Shah Farhat

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Background: Prenatal asphyxia or birth asphyxia is the medical situation resulting from a newborn infant that lasts long enough during the birth process to cause physical harm, usually to the brain. Human umbilical cord blood (UCB) is a well-established source of hematopoietic stem/progenitor cells (HSPCs) for allogeneic stem cell transplantation. These can be used clinically to care for children with malignant diseases. Low O2 can cause in proliferation and differentiation of stem cells. Method: the cord blood of 11 infants with 3-5 Apgar scores or need to cardiac pulmonary Resuscitation as an asphyxia group and ten normal infants with more than 8 Apgar scores as the normal group was collected, and after isolating hematopoietic stem cells, the cells were cultured in enriched media for 14 days to compare the numbers of colonies by microscope. Results: There was a significant difference in the number of RBC precursor colonies (red colonies) in cultured media with 107 cord blood hematopoietic stem cells of infants who were exposed to hypoxemia in two wells of palate. There was not a significant difference in the number of white cell colonies in the two groups in the two wells of the plate. Conclusion: Hypoxia in the perinatal period can cause the increase of hematopoietic stem cells of cord blood, special red precursor stem cells in vitro, like an increase of red blood cells in the body when exposed to low oxygen conditions. Thus, it will be usable.

Keywords: asphyxia, neonre, stem cell, red cell

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Authors: Alieh Farshbaf

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Introduction: Current anti-retroviral drugs have some restrictions for treatment of HIV-1 infection. The efficacy of retroviral drugs is not same in different infected patients and the virus rebound from latent reservoirs after stopping them. Recently, the engineering of stem cells and gene therapy provide new approaches to eliminate some drug problems by induction of resistance to HIV-1. Literature review: Up to now, AIDS-restriction genes (ARGs) were suitable candidate for gene and cell therapies, such as cc-chemokine receptor-5 (CCR5). In this manner, CCR5 provide effective cure in Berlin and Boston patients by inducing of HIV-1 resistance with allogeneic stem cell transplantation. It is showed that Zinc Finger Nuclease (ZFN) could induce HIV-1 resistance in stem cells of infected patients by homologous recombination or non-end joining mechanism and eliminate virus loading after returning the modified cells. Then, gene modification by HIV restriction factors, as TRIM5, introduced another gene candidate for HIV by interfering in infection process. These gene modifications/editing provided by stem cell futures that improve treatment in refractory disease such as HIV-1. Conclusion: Although stem cell transplantation has some complications, but in compare to retro-viral drugs demonstrated effective cure by elimination of virus loading. On the other hand, gene therapy is cost-effective for an infected patient than retroviral drugs payment in a person life-long. The results of umbilical cord blood stem cell transplantation showed that gene and cell therapy will be applied easier than previous treatment of AIDS with high efficacy.

Keywords: stem cell, AIDS, gene modification, cell engineering

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Authors: Sepideh Hassanpour Khodaei

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Background/Objectives: The purpose of this study is to evaluate the effect of mummy substances on two issues of proliferation and production of matrix protein synthesis in wound healing. Methods: The methodology used for this aim involves isolating mesenchymal stem cells and human fibroblasts procured at Pastor Institute, Iran. The cells were treated with mummy substances separately and co-cultured between ASCs and WJSCs, and fibroblasts. Proliferation was assessed by Ki67 method in monolayer conditions. Synthesis of components of extracellular matrix (ECM) such as collagen type I, type III, and fibronectin 1 (FN1) was determined by qPCR. Results: The effects of adipocyte stem cells (ASCs), Wharton Jelly Stem Cells (WJSCs), and Mummy material on fibroblast proliferation and migration were evaluated. The present finding underlined the importance of Mummy material, ASCs, and WJSCs in the proliferation and migration of fibroblast cells. Furthermore, the expression of collagen I, III, and FN1 was increased in the presence of the above material and cells. Conclusion: This study presented an effective in vitro method for the healing process. Hence, the prospect of utilizing Mummy material and stem cell-based therapies in wound healing as a therapeutic approach is promising.

Keywords: mummy material, wound healing, adipose tissue, Wharton’s jelly

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Authors: A. Reum Son, Jin Seon Kwon, Seung Hun Park, Hai Bang Lee, Moon Suk Kim

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These days, stem cell therapy is focused on for promising source of treatment in clinical human disease. As a supporter of stem cells, in situ-forming hydrogels with growth factors and cells appear to be a promising approach in tissue engineering. To examine osteogenic differentiation of hTMSCs which is one of mesenchymal stem cells in vivo in an injectable hydrogel, we use a methoxy polyethylene glycol-polycaprolactone blockcopolymer (MPEG-PCL) solution with osteogenic factors. We synthesized MPEG-PCL hydrogel and measured viscosity to check sol-gel transition. In order to demonstrate osteogenic ability of hTMSCs, we conducted in vitro osteogenesis experiment. Then, to confirm the cell cytotoxicity, we performed WST-1 with hTMSCs and MPEG-PCL. As the result of in vitro experiment, we implanted cell and hydrogel mixture into animal model and checked degree of osteogenesis with histological analysis and amount of expression genes. Through these experimental data, MPEG-PCL hydrogel has sol-gel transition in temperature change and is biocompatible with stem cells. In histological analysis and gene expression, hTMSCs are very good source of osteogenesis with hydrogel and will use it to tissue engineering as important treatment method. hTMSCs could be a good adult stem cell source for usability of isolation and high proliferation. When hTMSCs are used as cell therapy method with in situ-formed hydrogel, they may provide various benefits like a noninvasive alternative for bone tissue engineering applications.

Keywords: injectable hydrogel, stem cell, osteogenic differentiation, tissue engineering

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Authors: Hasan Mahmud Reza

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Extensive studies of the human umbilical cord, both basic and translational, over the last three decades have unveiled a plethora of information. The cord lining harbors at least two phenotypically different multipotent stem cells: mesenchymal stem cells (MSCs) and cord lining epithelial stem cells (CLECs). These cells exhibit a mixed genetic profiling of both embryonic and adult stem cells, hence display a broader stem features than cells from other sources. We have observed that umbilical cord-derived cells are immunologically privileged and non-tumorigenic by animal study. These cells are ethically acceptable, thus provides a significant advantage over other stem cells. The high proliferative capacity, viability, differentiation potential, and superior harvest of these cells have made them better candidates in comparison to contemporary adult stem cells. Following 30 replication cycles, these cells have been observed to retain their stemness, with their phenotype and karyotype intact. Transplantation of bioengineered CLEC sheets in limbal stem cell-deficient rabbit eyes resulted in regeneration of clear cornea with phenotypic expression of the normal cornea-specific epithelial cytokeratin markers. The striking features of low immunogenicity protecting self along with co-transplanted allografts from rejection largely define the transplantation potential of umbilical cord-derived stem cells.

Keywords: cord lining epithelial stem cells, mesenchymal stem cell, regenerative medicine, umbilical cord

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Authors: Huseyin Apdik, Aysegul Dogan, Selami Demirci, Ezgi Avsar Apdik, Fikrettin Sahin

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Mesenchymal stem cells (MSCs) are crucial cell types for bone maintenance and growth along with resident bone progenitor cells providing bone tissue integrity during osteogenesis and skeletal growth. Any deficiency in this regulation would result in vital bone diseases. Of those, osteoporosis, characterized by a reduction in bone mass and mineral density, is a critical skeletal disease for especially elderly people. The commonly used drugs for the osteoporosis treatment are bisphosphonates (BPs). The most prominent role of BPs is to prevent bone resorption arisen from high osteoclast activity. However, administrations of bisphosphonates may also cause bisphosphonate-induced osteonecrosis of the jaw (BIONJ). Up to the present, the researchers have proposed several circumstances for BIONJ. However, effects of long-term and/or high dose usage of BPs on stem cell’s proliferation, survival, differentiation or maintenance capacity have not been evaluated yet. The present study will be held to; figure out BPs’ effects on MSCs in vitro in the aspect of cell proliferation and toxicity, migration, angiogenic activity, lineage specific gene and protein expression levels, mesenchymal stem cell properties and potential signaling pathways affected by BP treatment. Firstly, mesenchymal stem cell characteristics of Dental Pulp Stem Cells (DPSCs) and Periodontal Ligament Stem Cells (PDLSCs) were proved using flow cytometry analysis. Cell viability analysis was completed to determine the cytotoxic effects of BPs (Zoledronate (Zol), Alendronate (Ale) and Risedronate (Ris)) on DPSCs and PDLSCs by the 3-(4,5-di-methyl-thiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfo-phenyl)-2H-tetrazolium (MTS) assay. Non-toxic concentrations of BPs were determined at 24 h under growth condition, and at 21 days under osteogenic differentiation condition for both cells. The scratch assay was performed to evaluate their migration capacity under the usage of determined of BPs concentrations at 24 h. The results revealed that while the scratch closure is 70% in the control group for DPSCs, it was 57%, 66% and 66% in Zol, Ale and Ris groups, respectively. For PDLSs, while wound closure is 71% in control group, it was 65%, 66% and 66% in Zol, Ale and Ris groups, respectively. As future experiments, tube formation assay and aortic ring assay will be done to determinate angiogenesis abilities of DPSCs and PDLSCs treated with BPs. Expression levels of osteogenic differentiation marker genes involved in bone development will be determined using real time-polymerase change reaction (RT-PCR) assay and expression profiles of important proteins involved in osteogenesis will be evaluated using western blotting assay for osteogenically differentiated MSCs treated with or without BPs. In addition to these, von Kossa staining will be performed to measure calcium mineralization status of MSCs.

Keywords: bisphosphonates, bisphosphonate-induced osteonecrosis of the jaw, mesenchymal stem cells, osteogenesis

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Authors: Norimichi Nagano, Yoshio Hirano, Tsutomu Yasukawa

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Mesenchymal stem cells are thought to confer neuroprotection, facilitate tissue regeneration and exert their effects on retinal degenerative diseases, however, adverse events such as proliferative vitreoretinopathy and preretinal membrane disease associated with cell suspension transplantation have also been reported. We have recently developed human (allogeneic) adipose tissue-derived mesenchymal stem cell (adMSC) sheets through our proprietary sheet transformation technique, which could potentially mitigate these adverse events. To clarify the properties of our adMSC sheets named PAL-222, we performed in vitro studies such as viability testing, cytokine secretions by ELISA, immunohistochemical study, and migration assay. The viability of the cells exceeded 70%. Vascular Endothelial Growth Factor (VEGF) and Pigment Epithelium-Derived Factor (PEDF), which are quite important cytokines for the retinal area, were observed. PAL-222 expressed type I collagen, a strength marker, type IV collagen, a marker of the basement membrane, and elastin, an elasticity marker. Finally, the migration assay was performed and showed negative, which means that PAL-222 is stably kept in the topical area and does not come to pieces. Next, to evaluate the efficacy in vivo, we transplanted PAL-222 into the subretinal space of the eye of Royal College of Surgeons rats with congenital retinal degeneration and assessed it for three weeks after transplantation. We confirmed that PAL-222 suppressed the decrease in the thickness of the outer nuclear layer, which means that the photoreceptor protective effect treated with PAL-222 was significantly higher than that in the sham group. (p < 0.01). This finding demonstrates that PAL-222 showed their retinoprotective effect in a model of congenital retinal degeneration. As the study suggested the efficacy of PAL-222 in both in vitro and in vivo studies, we are presently engaged in clinical trials of PAL-222 for myopic chorioretinal atrophy, which is one of the retinal degenerative diseases, for the purpose of regenerative medicine.

Keywords: cell sheet, clinical trial, mesenchymal stem cell, myopic chorioretinal atrophy

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Authors: Ainur Mukhambetova, Miras Karzhauov, Vyacheslav Ogay

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Introduction: Mesenchymal stem cells (MSCs) have the capacity to be differentiated into several cell lineages and are a promising source for cell therapy and tissue engineering. However, currently most MSCs culturing protocols use media supplemented with fetal bovine serum (FBS), which limits their application in clinic due to the possibility of zoonotic infections, contamination and immunological reactions. Consequently, formulating effective serum free culture medium becomes one of the important problems in contemporary cell biotechnology. Objectives: The aim of this study was to define an optimal serum-free medium for culturing of periosteum derived MSCs. Materials and methods: The MSCs were extracted from human periosteum and transferred to the culture flasks pretreated with CELLstart™. Immunophenotypic characterization, proliferation and in vitro differentiation of cells grown on STEM PRO® MSC SFM were compared to the cells cultured in the standard FBS containing media. Chromosome analysis and flow cytometry were also performed. Results: We have shown that cells were grown on STEM PRO® MSC SFM retained all the morphological, immunophenotypic (CD73, CD90, CD105, vimentin and Stro-1) and cell differentiation characteristics specific to MSCs. Chromosome analysis indicated no anomalies in the chromosome structure. Flow cytometry showed a high expression of cell adhesion molecules CD44 (98,8%), CD90 (97,4%), CD105 (99,1%). In addition, we have shown that cell is grown on STEM PRO® MSC SFM have higher proliferation capacity compared to cell expanded on standard FBS containing the medium. Conclusion: We have shown that STEM PRO® MSC SFM is optimal for culturing periosteum derived human MSCs which subsequently can be safely used in cell therapy.

Keywords: cell technologies, periosteum-derived MSCs, regenerative medicine, serum-free medium

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Authors: Remi Safi, Aline Hamade, Najat Bteich, Jamal El Saghir, Mona Diab Assaf, Marwan El-Sabban, Fadia Najjar

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Estrogen is considered a risk factor for breast cancer since it promotes breast-cell proliferation. The jaesckeanadiol-3-p-hydroxyphenylpropanoate, a hemi-synthetic analogue of the natural phytoestrogen ferutinin (jaesckeanadiol-p-hydroxybenzoate), is designed to be devoid of estrogenic activity. This analogue induces a cytotoxic effect 30 times higher than that of ferutinin towards MCF-7 breast cancer cell line. We compared these two compounds with respect to their effect on proliferation, cell cycle distribution and cancer stem-like cells in the MCF-7 cell line. Treatment with ferutinin (30 μM) and its analogue (1 μM) produced a significant accumulation of cells at the pre G0/G1 cell cycle phase and triggered apoptosis. Importantly, this compound retains its anti-proliferative activity against breast cancer stem/progenitor cells that are naturally insensitive to ferutinin at the same dose. These results position ferutinin analogue as an effective compound inhibiting the proliferation of estrogen-dependent breast cancer cells and consistently targeting their stem-like cells.

Keywords: ferutinin, hemi-synthetic analogue, breast cancer, estrogen, stem/progenitor cells

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Authors: Melanie Macgregor-Ramiasa, Isabel Hopp, Patricia Murray, Krasimir Vasilev

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Stem cells based therapies are one of the greatest promises of new-age medicine due to their potential to help curing most dreaded conditions such as cancer, diabetes and even auto-immune disease. However, establishing suitable in vitro culture materials allowing to control the fate of stem cells remain a challenge. Amongst the factor influencing stem cell behavior, substrate chemistry and nanotopogaphy are particularly critical. In this work, we used plasma assisted surface modification methods to produce model substrates with tailored nanotopography and controlled chemistry. Three different sizes of gold nanoparticles were bound to amine rich plasma polymer layers to produce homogeneous and gradient surface nanotopographies. The outer chemistry of the substrate was kept constant for all substrates by depositing a thin layer of our patented biocompatible polyoxazoline plasma polymer on top of the nanofeatures. For the first time, protein adsorption and stem cell behaviour (mouse kidney stem cells and mesenchymal stem cells) were evaluated on nanorough plasma deposited polyoxazoline thin films. Compared to other nitrogen rich coatings, polyoxazoline plasma polymer supports the covalent binding of proteins. Moderate surface nanoroughness, in both size and density, triggers cell proliferation. In association with polyoxazoline coating, cell proliferation is further enhanced on nanorough substrates. Results are discussed in term of substrates wetting properties. These findings provide valuable insights on the mechanisms governing the interactions between stem cells and their growth support.

Keywords: nanotopography, stem cells, differentiation, plasma polymer, oxazoline, gold nanoparticles

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Authors: Loredana G. Marcu, David Marcu, Sanda M. Filip

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Advanced head and neck cancers are aggressive tumours, which require aggressive treatment. Treatment efficiency is often hindered by cancer cell repopulation during radiotherapy, which is due to various mechanisms triggered by the loss of tumour cells and involves both stem and differentiated cells. The aim of the current paper is to present in silico simulations of radiotherapy schedules on a virtual head and neck tumour grown with biologically realistic kinetic parameters. Using the linear quadratic formalism of cell survival after radiotherapy, altered fractionation schedules employing various treatment breaks for normal tissue recovery are simulated and repopulation mechanism implemented in order to evaluate the impact of various cancer cell contribution on tumour behaviour during irradiation. The model has shown that the timing of treatment breaks is an important factor influencing tumour control in rapidly proliferating tissues such as squamous cell carcinomas of the head and neck. Furthermore, not only stem cells but also differentiated cells, via the mechanism of abortive division, can contribute to malignant cell repopulation during treatment.

Keywords: radiation, tumour repopulation, squamous cell carcinoma, stem cell

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Authors: Parcha Sreenivasa Rao, P. Lakshmi

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Diabetes Mellitus is metabolic disorder, characterized by high glucose levels in the blood due to one of the reason i.e., the death of β cells. The lack of β cells leads to the reduced insulin levels. The β cell death generally occurs due to apoptosis induced by the several cytokines. IL-1β, IFN- ϒ and TNF –α cytokines that are generally cause apoptosis to the β cell. The nutrient based apoptosis is generally seen with high glucose and free fatty acids. It is also noted that the β cell death triggered by Fas ligand and its receptor Fas at the surface of the activated CD8+ T- lymphocytes. Reports also reveal that the β cell apoptosis is under control of the transcription factors NF-kB and STAT- 1. The arresting or opposing of the β cell apoptosis can be overcome by the different growth factors like GLP-1, growth hormone, prolactin, VEGF, Dipeptidyl peptidase-4, Vildagliptin, suberoylanilidehydroxamic acid, trichistatin-A, XIAP, Bcl-2, FGF-21. Present investigation explains antiapoptotic property of the β cells derived from the mesenchymal stem cells of umbilical cord.

Keywords: stem cells, umblical cord, diabetes, apoptosis

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Authors: Balakrishna Shetty

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Introduction: Stem Cell Imaging is a challenging field since the advent of Stem Cell treatment in humans. Series of research on tagging and tracking the stem cells has not been very effective. The present study is an effort by the authors to track the stem cells injected into calf muscles by Magnetic Resonance Diffusion Weighted Imaging. Materials and methods: Stem Cell injection deep into the calf muscles of patients with peripheral vascular disease is one of the recent treatment modalities followed in our institution. 5 patients who underwent deep intramuscular injection of stem cells as treatment were included for this study. Pre and two hours Post injection MRI of bilateral calf regions was done using 1.5 T Philips Achieva, 16 channel system using 16 channel torso coils. Axial STIR, Axial Diffusion weighted images with b=0 and b=1000 values with back ground suppression (DWIBS sequence of Philips MR Imaging Systems) were obtained at 5 mm interval covering the entire calf. The invert images were obtained for better visualization. 120ml of autologous bone marrow derived stem cells were processed and enriched under c-GMP conditions and reduced to 40ml solution containing mixture of above stem cells. Approximately 40 to 50 injections, each containing 0.75ml of processed stem cells, was injected with marked grids over the calf region. Around 40 injections, each of 1ml normal saline, is injected into contralateral leg as control. Results: Significant Diffusion hyper intensity is noted at the site of injected stem cells. No hyper intensity noted before the injection and also in the control side where saline was injected conclusion: This is one of the earliest studies in literature showing diffusion hyper intensity in intramuscularly injected stem cells. The advantages and deficiencies in this study will be discussed during the presentation.

Keywords: stem cells, imaging, DWI, peripheral vascular disease

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Authors: Dinara Ikramova, Karin A. Hing, Simon C. F. Rawlinson

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Silicate-substituted hydroxyapatite (SiHA) has been shown to enhance bone regeneration in vivo compared with phase pure stoichiometric hydroxyapatite. Evidence suggests that substrate chemistry dependent formation of a permissive protein layer on the surface of synthetic bone graft substitute materials is key for bioactivity and cell attachment. However, little information is available on whether the substrate chemistry may affect cell migration and recruitment. The aim of this study is to investigate whether or not human Mesenchymal Stem Cells (hMSCs) exhibit a chemotactic response to SiHA porous granules and if it can be linked to either the ion exchange or protein sequestering and enrichment on the surface of the material. 150mg of SiHA granules with 80% total porosity and 20% strut porosity were incubated in 1ml of either Serum Free Media (SFM) or 10% Serum Containing Media (SCM) under static cell culture conditions (37°C, 5% CO2) in absence of cells. Protein sequestering and exchange of calcium, phosphate and silicate ions were analysed at 0.5, 1, 2, 4, 8, 16 and 24 hours with n=12 per time point. Migration of hMSCs in the presence of 150mg of SiHA granules was assessed over 24 hours using a modified transwell migration system in either SFM or SCM (n=6) with 30% serum containing media acting as a positive control. At 24 hours protein sequestering and ionic exchange were analysed, and the number of cells was quantified using a high throughput confocal microscope (IN Cell Analyser 6000). In acellular condition, both calcium and phosphate ion concentrations in media showed a decrease at 24 hours which was greater in SFM than in SCM. This suggests possible formation and precipitation of a bone like apatite on the surface of SiHA. Reduction in this activity observed in SCM indicates that the presence of serum proteins is interfering with the ion exchange at the material and media interface. Adsorbed protein levels showed fluctuation over time followed by sharp decrease at 24 hours, suggesting a possible protein rearrangement on the surface of the material. The ion analysis performed on SFM and SCM after 24-hour incubation with cells in the presence of granules showed a greater reduction in phosphate concentration in both SFM and SCM compared to phosphate levels in acellular condition. Silicate concentration in SCM increased from 1.6mM (absence of cells) to 5.1mM (presence of cells). This indicates that the cells are promoting the uptake of phosphate and release of silicate ions. No significant change was seen in levels of adsorbed proteins in the presence and absence of cells. Further analysis is required to determine whether the species of these proteins change over time. The analysis of cell migration after 24-hour incubation showed more cells migrating towards the granules, 12.7% in SFM and 8.3% in SCM, than in positive control, 4.5% in SFM and 3.6% in SCM respectively. These results suggest that SiHA has a chemotactic activity independent of serum proteins. A property which has not previously been demonstrated for a synthetic bone graft material.

Keywords: cell migration, hMSCs, SiHA, transwell migration system

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Authors: Laila Montaser, Hala Gabr, Maha El-Bassuony, Gehan Tawfeek

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Orthotropic liver transplantation (OLT) is the final procedure of both end stage and metabolic liver diseases. Hepatocyte transplantation is an alternative for OLT, but the sources of hepatocytes are limited. Bone marrow mesenchymal stem cells (BM-MSCs) can differentiate into hepatocyte-like cells and are a potential alternative source for hepatocytes. The MSCs from bone marrow are a promising target population as they are capable of differentiating along multiple lineages and, at least in vitro, have significant expansion capability. MSCs from bone marrow may have the potential to differentiate in vitro and in vivo into hepatocytes. Our study examined whether mesenchymal stem cells (MSCs), which are stem cells originated from human bone marrow, are able to differentiate into functional hepatocyte-like cells in vitro. Our aim was to investigate the differentiation potential of BM-MSCs into hepatocyte-like cells. Adult stem cell therapy could solve the problem of degenerative disorders, including liver disease.

Keywords: bone marrow, differentiation, hepatocyte, stem cells

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Authors: Yu-Chen Hu

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Gene editing by CRISPR and gene regulation by microRNA or CRISPR activation have dramatically changed the way to manipulate cellular gene expression and cell fate. In recent years, various gene editing and gene manipulation technologies have been applied to control stem cell differentiation to enhance tissue regeneration. This research will focus on how to develop CRISPR, CRISPR activation (CRISPRa), CRISPR inhibition (CRISPRi), as well as bi-directional CRISPR-AI gene regulation technologies to control cell differentiation and bone regeneration. Moreover, in this study, CRISPR/Cas13d-mediated RNA editng for miRNA editing and bone regeneration will be discussed.

Keywords: gene therapy, bone regeneration, stem cell, CRISPR, gene regulation

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Authors: Ognen Kostovski, Svetozar Antovic, Rubens Jovanovic, Irena Kostovska, Nikola Jankulovski

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Introduction:Colorectal carcinoma (CRC) is one of the most common malignancies in the world. The cancer stem cell (CSC) markers are associated with aggressive cancer types and poor prognosis. The aim of study was to determine whether the expression of colorectal cancer stem cell markers CD133 and CD44 could be significant in prediction of clinically aggressive form of CRC. Materials and methods: Our study included ninety patients (n=90) with CRC. Patients were divided into two subgroups: with metatstatic CRC and non-metastatic CRC. Tumor samples were analyzed with standard histopathological methods, than was performed immunohistochemical analysis with monoclonal antibodies against CD133 and CD44 stem cell markers. Results: High coexpression of CD133 and CD44 was observed in 71.4% of patients with metastatic disease, compared to 37.9% in patients without metastases. Discordant expression of both markers was found in 8% of the subgroup with metastatic CRC, and in 13.4% of the subgroup without metastatic CRC. Statistical analyses showed a significant association of increased expression of CD133 and CD44 with the disease stage, T - category and N - nodal status. With multiple regression analysis the stage of disease was designate as a factor with the greatest statistically significant influence on expression of CD133 (p <0.0001) and CD44 (p <0.0001). Conclusion: Our results suggest that the coexpression of CD133 and CD44 have an important role in prediction of clinically aggressive form of CRC. Both stem cell markers can be routinely implemented in standard pathohistological diagnostics and can be useful markers for pre-therapeutic oncology screening.

Keywords: colorectal carcinoma, stem cells, CD133+, CD44+

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Authors: Naser Shagerdi Esmaeli, Mohsen Hamidpour, Parisa Hasankhani Tehrani

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Background And Objective: Haematopoietic stem cell transplant is a potentially curative treatment option in various benign and malignant haematological diseases. Patients undergoing stem cell transplant procedure require blood transfusion on a daily basis. Currently, there is paucity of data from developing countries on transfusion practices. This audit was undertaken to determine the consumption of packed red blood cells (PRBCs) transfusion in the bone marrow transplant unit of the Shahid Ghazi Tabatabaei Hospital, Tabriz, Iran. Subjects And Methods: A retrospective audit was conducted for packed red cell transfusion ordering practice over a period from March 2017 to march 2018. All consecutive patients admitted for stem cell transplant procedure for various underlying diseases were included. Outcome measures used in this study were (i) cross match to transfusion (C: T) ratio and (ii) transfusion trigger. Results: During the study period, n=13 patients underwent a haematopoietic stem cell transplant. There were n=10 males and n=3 females. One patient was less than 15 years of age, while rests were adults. Median age±SD was 26.5±14.5 years (12∼54 years). The underlying diagnosis included Aplastic anemia (n=4), Thalassemia major (n=1), Multiple Myeloma (n=3), Acute leukemia (n=3), Hodgkin's lymphoma (n=1), PRCA (n=1). Grand total consumption of PRBCs during the study period was 204, while 258 products were crossmatch. The C:T ratio was 1.26. The transfusion trigger was Hb level of less than 8 gr/dl. Conclusion: The results of our BMT unit indicate that the C:T ratio and transfusion trigger is comparable to the international criteria and pioneer country in BMT transplantation. Also, we hope that our blood consumption become less than it is now.

Keywords: blood consumption, C: T ratio, PRBCs, stem cell transplant, tabriz, Iran

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Authors: Natasha Petrovska, Mirjana Pavlovic, Maria M. Larrondo-Petrie

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Neural stem cells (NSCs) are multi-potent, self-renewing cells that generate new neurons. Three subtypes of NSCs can be separated regarding the stages of NSC lineage: quiescent neural stem cells (qNSCs), activated neural stem cells (aNSCs) and neural progenitor cells (NPCs), but their gene expression signatures are not utterly understood yet. Single-cell examinations have started to elucidate the complex structure of NSC populations. Nevertheless, there is a lack of thorough molecular interpretation of the NSC lineage heterogeneity and an increasing need for tools to analyze and improve the efficiency and correctness of single-cell sequencing data. Feature selection and ordering can identify and classify the gene expression signatures of these subtypes and can discover novel subpopulations during the NSCs activation and differentiation processes. The aim here is to review the implementation of the feature selection technique on NSC subtypes and the classification techniques that have been used for the identification of gene expression signatures.

Keywords: feature selection, feature similarity, neural stem cells, genes, feature selection methods

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