Culture Of Progenitor Cells Research Articles

Effects of ischemia-reperfusion on endothelial progenitor cell function and iNOS, eNOS expression

Objective To explore the effects of ischemia and ischemia reperfusion on the proliferation, apoptosis, migration ability, inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression of endothelial progenitor cells (EPCs). Methods Collection of peripheral blood from volunteers and culture of endothelial progenitor cells in vitro. The cells were divided into three groups: control group, hypoxia group and hypoxia reoxygenation group. Methyl thiazolyl tetrazolium (MTT) assay was used to detect cell proliferation. Transwell chamber method was used to detect cell migration. Cell apoptosis was detected by flow cytometry. Western blot was used to detect iNOS and eNOS expressions. Results A confocal microscope was used to observe the basic adherence of the cells to the wall for about 3 days, and the area became larger. After 7 d of single nucleus cell culture, the growth of colony-like pattern was more than that of spindle. The cell counts of the three groups in the microscope were (1.83±0.92), (5.07±0.84), (2.11±0.74). Compared with the control group (0.24±0.04), the hypoxia group (0.62±0.06) could promote EPCs proliferation, and the difference was statistically significant (t=12.142, P 0.05). The number of cell migration in the hypoxia group (18.28±2.05) and hypoxic complex oxygen group (14.08±2.11) was not statistically significant compared with the control group (15.14±1.25) (P>0.05). The apoptosis rate in hypoxia group (34.57±0.42)% and hypoxia reoxygenation group (41.08±0.44)% was significantly higher than that in control group (24.83±0.38)% (χ2=13.427, 15.084, P<0.05). The apoptosis rate of hypoxia reoxygenation group was significantly higher than that of hypoxia group (χ2=9.657, P<0.05). The expression of iNOS in hypoxia group and hypoxia reoxygenation group was significantly higher than that in control group, and the difference was statistically significant (P<0.05). Conclusions Ischemia could promote the proliferation of EPCs, and increase the expression of iNOS, but the expression of EPCs was down-regulated after reperfusion. Key words: Reperfusion/AE; Endothelium/PA; Stem cells/PA; Nitric oxide synthase/ME

Automated Microfluidic System for Dynamic Stimulation and Tracking of Single Cells.

Dynamic environments determine cell fate decisions and function. Understanding the relationship between extrinsic signals on cellular responses and cell fate requires the ability to dynamically change environmental inputs in vitro, while continuously observing individual cells over extended periods of time. This is challenging for nonadherent cells, such as hematopoietic stem and progenitor cells, because media flow displaces and disturbs such cells, preventing culture and tracking of single cells. Here, we present a programmable microfluidic system designed for the long-term culture and time-lapse imaging of nonadherent cells in dynamically changing cell culture conditions without losing track of individual cells. The dynamic, valve-controlled design permits targeted seeding of cells in up to 48 independently controlled culture chambers, each providing sufficient space for long-term cell colony expansion. Diffusion-based media exchange occurs rapidly and minimizes displacement of cells and eliminates shear stress. The chip was successfully tested with long-term culture and tracking of primary hematopoietic stem and progenitor cells, and murine embryonic stem cells. This system will have important applications to analyze dynamic signaling inputs controlling fate choices.

Abstract 2411: Reactive Oxygen Species regulates tumor stem cell survival in medulloblastoma via mitochondrial biogenesis

Abstract Sonic hedgehog (Shh) signaling is closely coupled with the bioenergetics of medulloblastoma, the most common malignant pediatric solid tumor. We have previously reported that Shh causes deregulation of mitochondrial biogenesis by suppressing mitofusins. This led to fragmented mitochondria in vitro in primary cultures of mouse cerebellar progenitor cells, putative cells-of-origin of the SHH molecular subclass of medulloblastoma. This phenomenon was also observed in electron micrographs of SmoA1 mouse medulloblastomas in vivo. Ectopic expression of mitofusins in both proliferating progenitor cells and primary mouse medulloblastoma cell cultures restored mitochondrial fusion and led to a non-proliferative phenotype (1). We have observed high levels of Reactive Oxygen Species (ROS) in SmoA1 mouse medulloblastoma cells in vitro. When total ROS in primary cultures of mouse medulloblastoma cells was scavenged by N-Acetyl Cytosine (NAC) treatment, the mitochondria regained their fused morphology, leading to a reduction in cell proliferation. NAC treatment of organotypic cerebellar slice cultures implanted with SmoA1 neurospheres also resulted in a reduction in proliferation as well as impaired spread of tumor cells within normal cerebellar tissue. Medulloblastoma tumor repopulating cells, which are responsible for radiation resistance and tumor recurrence, are primarily located in the perivascular niche (PVN) of Medulloblastoma (2). We therefore focused our attention on the role played by ROS in regulating mitochondrial structure of tumor repopulating cells, to decipher if this affects the survival of these cells. Indeed we have observed that NAC treatment combined with irradiation restores mitochondrial morphology, leading to enhanced cell death within the PVN of Medulloblastoma. We further carried out gene expression analysis of ROS-regulating genes in the Shh subgroup of Medulloblastoma. NADPH Oxidase 4 (NOX4), which is a known producer of cytoplasmic ROS in cells (3), was found to be upregulated as compared to NOX1 and 3. This elevation was observed across all subgroups of medulloblastoma, but was most evident in the Shh subgroup. We have also found that NOX4 co-localizes in the PVN of medulloblastoma. Treatment with Apocynin, a NOX2/4 inhibitor, led to enhanced cell death in the PVN, similar to what we observed after NAC treatment. Taken together, our results show that anti-oxidant treatment restores the fused morphology of mitochondria in the PVN and leads the cells to apoptosis, thereby preventing tumor recurrence post-irradiation, and they implicate Nox4 as a potential therapeutic target. 1. Malhotra A, Dey A, Prasad N, Kenney AM. 2015. Mol Cancer Res 2. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC. 2008. Genes Dev 22: 436-48 3. Bedard K, Krause KH. 2007. Physiol Rev 87: 245-313 Citation Format: Anshu Malhotra, Abhinav Dey, Anna M. Kenney. Reactive Oxygen Species regulates tumor stem cell survival in medulloblastoma via mitochondrial biogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2411.

AN AMYLOID-BETA-DERIVED PEPTIDE TRANSFORMS RAT GLIAL PROGENITOR CELLS INTO MICROGLIA

Septapeptides (“septas”), previously identified as meningitis-specific antigens, defined by a rubella virus monoclonal antibody, were found in human Monocyte Chemoattractant Protein (hMCP-1) and on the surface of meningitis-causing bacteria, viruses and spirochetes.1-3 Some bacterial septas were tested for Ca+2 mobilization through receptor-associated heterotrimeric G-protein binding on THP-1 cells, progenitor cells of circulating peripheral macrophages. Certain of the free septas acted on their own as mild agonists of Ca+2 mobilization. Their signal transduction activity may be mediated through a single (or a single class) of receptor, but the data do not link this with the MCP-1 receptor on THP-1 cells. These data support the notion that (1) infectious organisms may conserve and employ these sequences in order to facilitate their transport through the BBB to infect the CNS and (2) the hypothesis that the septas represent muteins of the MCP-1 active site for monocyte stem cell activation to macrophages. Other MCP muteins have since been identified in the AD-associated agents, amyloid beta and prions, as well as in viruses like HIV, known to establish chronic infections in the CNS. Rat glial progenitor cells in tissue culture 4-6 were used to test for hMCP-1 activity in septas derived from amyloid beta. Nanomolar concentrations of the amyloid beta septa 13HHQKLVF19 were found to transform more than 60% of rat glial progenitor cells into mature microglia in tissue culture7.8. These data support the hypothesis that an amyloid beta-derived septa may serve to activate stem cells to increase the number of microglia available to combat chronic infection in the CNS. 1VanAlstyne & Sharma. April 23, 1996. US Patent No. 5,510,264. 2VanAlstyne & Sharma. Sept. 17, 1996. US Patent No. 5,556,757. 3Van Alstyne & Sharma. Dec. 6, 2011. US Patent No. 8,071,102. 4Singh and Van Alstyne. 1978.Brain Res. 155:418-421. 5Pope and Van Alstyne. 1981.Virology 113(2):776-780. 6Van Alstyne and Paty. 1983.Virology 124:173-180 7Van Alstyne, Jan. 4, 2018. US Patent Application 62/613,621. 8Van Alstyne. June 20, 2018 Poster(3035) presentation at Keystone Symposia Conference “Advances in Neurodegenerative Disease Research and Therapy”. Session Z3.

Isolation, culture and identification of bone marrow-derived endothelial progenitor cells in mice

Objective To explore a practical and feasible method for isolation, culture and identification of mouse bone marrow endothelial progenitor cells(EPCs). Methods Bone marrow-derived mononuclear cells isolated by density gradient centrifugation were cultured in endothelial cell growth medium-2 MV medium. Growth and morphological changes of the cells were observed under inverted microscopy. Cell proliferation was observed by cell counting kit-8 assay. Surface markers of the EPCs were detected by flow cytometry. Angiogenic tube formation was determined by Matrigel tube formation assay. Fluores cein isothiocyanat e-ulex europaeus agglutinin-1(FITC-UEA-1) binding and Dil-Ac-LDL uptake capabilities were observed by fluorescent microscopy. Results In the early stage, the cells were round and spindle-shaped after induced culture for 4 days. After 7 days, the cells grew in colony arrangement and gradually increased in number. After 14 days, the cells were differently shaped, such as short shuttle and triangle. After 21 days, the typical paving stone appearance of the cells was observed. The cells were positive for endothelial markers in flow cytometry: CD34+ (84.3%), vascular endothelial growth factor receptor 2+ (74.1%), but CD45+ (4.04%). The cells were capable of forming capillary-like tubes, up-taking Dil-Ac-LDL and binding FITC-UEA-1 in Matrigels. Conclusions A reliable method for isolation, culture and identification of mouse bone marrow EPCs may be improved on the basis of previous experiences. Since the EPCs obtained by this method may be capable of good proliferation, large in number, and stable in biological characteristics, they can serve as ideal seed cells for related subsequent studies. Key words: Bone marrow; Cell culture techniques; Monocytes; Endothelial progenitor cells; Cell identification

Isolation and culture of human alveolar epithelial progenitor cells

Isolation and culture of human alveolar epithelial progenitor cells

Pyruvate Kinase M2 Increases Angiogenesis, Neurogenesis, and Functional Recovery Mediated by Upregulation of STAT3 and Focal Adhesion Kinase Activities After Ischemic Stroke in Adult Mice.

Ischemic stroke remains a serious threat to human life. Generation of neuronal and vascular cells is an endogenous regenerative mechanism in the adult brain, which may contribute to tissue repair after stroke. However, the regenerative activity is typically insufficient for significant therapeutic effects after brain injuries. Pyruvate kinase isoform M2 (PKM2) is a key regulator for energy metabolism. PKM2 also has nonmetabolic roles involving regulations of gene expression, cell proliferation, and migration in cancer cells as well as noncancerous cells. In a focal ischemic stroke mouse model, recombinant PKM2 (rPKM2) administration (160ng/kg, intranasal delivery) at 1 h after stroke showed the significant effect of a reduced infarct volume of more the 60%. Delayed treatment of rPKM2, however, lost the acute neuroprotective effect. We then tested a novel hypothesis that delayed treatment of PKM2 might show proregenerative effects for long-term functional recovery and this chronic action could be mediated by its downstream STAT3 signaling. rPKM2 (160ng/kg) was delivered to the brain using noninvasive intranasal administration 24h after the stroke and repeated every other day. Western blot analysis revealed that, 7days after the stroke, the levels of PKM2 and phosphorylated STAT3 and the expression of angiogenic factors VEGF, Ang-1, and Tie-2 in the peri-infarct region were significantly increased in the rPKM2 treatment group compared with those of the stroke vehicle group. To label proliferating cells, 5-bromo-2'-deoxyuridine (BrdU, 50mg/kg, i.p.) was injected every day starting 3days after stroke. At 14days after stroke, immunohistochemistry showed that rPKM2 increased cell homing of doublecortin (DCX)-positive neuroblasts to the ischemic cortex. In neural progenitor cell (NPC) cultures, rPKM2 (0.4-4nM) increased the expression of integrin β1 and the activation/phosphorylation of focal adhesion kinase (FAK). A mediator role of FAK in PKM2-promoted cell migration was verified in FAK-knockout fibroblast cultures. In the peri-infarct region of the brain, increased numbers of Glut-1/BrdU and NeuN/BrdU double-positive cells indicated enhanced angiogenesis and neurogenesis, respectively, compared to stroke vehicle mice. Using Laser Doppler imaging, we observed better recovery of the local blood flow in the peri-infarct region of rPKM2-treated mice 14days after stroke. Meanwhile, rPKM2 improved the sensorimotor functional recovery measured by the adhesive removal test. Inhibiting the STAT3 phosphorylation/activation by the STAT3 inhibitor, BP-1-102 (3mg/kg/day, o.g.), abolished all beneficial effects of rPKM2 in the stroke mice. Taken together, this investigation provides the first evidence demonstrating that early treatment of rPKM2 shows an acute neuroprotective effect against ischemic brain damage, whereas delayed rPKM2 treatment promotes regenerative activities in the poststroke brain leading to better functional recovery. The underlying mechanism involves activation of the STAT3 and FAK signals in the poststroke brain.

Porous microspheres support mesenchymal progenitor cell ingrowth and stimulate angiogenesis.

Porous microspheres have the potential for use as injectable bone fillers to obviate the need for open surgery. Successful bone fillers must be able to support vascularisation since tissue engineering scaffolds often cease functioning soon after implantation due to a failure to vascularise rapidly. Here, we test the angiogenic potential of a tissue engineered bone filler based on a photocurable acrylate-based high internal phase emulsion (HIPE). Highly porous microspheres were fabricated via two processes, which were compared. One was taken forward and investigated for its ability to support human mesenchymal progenitor cells and angiogenesis in a chorioallantoic membrane (CAM) assay. Porous microspheres with either a narrow or broad size distribution were prepared via a T-junction microfluidic device or by a controlled stirred-tank reactor of the HIPE water in oil in water (w/o/w), respectively. Culture of human embryonic stem cell-derived mesenchymal progenitor (hES-MP) cells showed proliferation over 11 days and formation of cell-microsphere aggregates. In-vitro, hES-MP cells were found to migrate into microspheres through their surface pores over time. The presence of osteoblasts, differentiated from the hES-MP cells, was evidenced through the presence of collagen and calcium after 30 days. Microspheres pre-cultured with cells were implanted into CAM for 7 days and compared with control microspheres without pre-cultured cells. The hES-MP seeded microspheres supported greater angiogenesis, as measured by the number of blood vessels and bifurcations, while the empty scaffolds attracted host chick cell ingrowth. This investigation shows that controlled fabrication of porous microspheres has the potential to create an angiogenic, bone filling material for use as a cell delivery vehicle.

Anti-aging Properties of Conditioned Media of Epidermal Progenitor Cells Derived from Mesenchymal Stem Cells.

IntroductionReduced number and activities of epidermal stem cells are related to the features of photoaged skin. It was reported that conditioned media from various stem cell cultures are capable of improving the signs of cutaneous aging. This work was performed to establish epidermal progenitor cells derived from mesenchymal stem cells, and to evaluate the anti-aging efficacy of its conditioned media.MethodsEpidermal progenitor cell culture was established by differentiation from mesenchymal stem cells, and its conditioned medium (EPC-CM) was prepared. Normal human dermal fibroblasts were exposed to hydrogen peroxide and the protective effects of EPC-CM were investigated, monitoring intracellular reactive oxygen species (ROS), cellular defense enzymes, collagen biosynthesis, and mitogen-associated protein kinase (MAPK) signaling. Anti-aging efficacy of cosmetic essence (5% EPC-CM) was evaluated by a clinical test with 25 Korean women aged between 29 and 69.ResultsHydrogen peroxide hindered proliferation of fibroblasts and increased the levels of intracellular ROS. Pretreatment of EPC-CM protected fibroblasts from oxidative stress as shown by accelerated proliferation and reduced ROS generation. EPC-CM effectively prevented hydrogen peroxide-induced alterations of the activities, as well as mRNA and protein levels, of antioxidative enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase. Reduced type I collagen biosynthesis and stimulated phosphorylation of MAPK signaling proteins, induced by oxidative damage, were also prevented by EPC-CM. In clinical study, wrinkle, depression, and skin texture were improved by the topical application of a formulation containing 5% EPC-CM within 4 weeks.ConclusionEpidermal progenitor cell culture was established, and its conditioned medium was developed for anti-aging therapy. EPC-CM improved signs of skin aging in clinical study, possibly via activation of cellular the defense system, as supported by in vitro results.Electronic supplementary materialThe online version of this article (10.1007/s13555-018-0229-2) contains supplementary material, which is available to authorized users.

Vitamin D Improves Neurogenesis and Cognition in a Mouse Model of Alzheimer\u2019s Disease

The impairment of hippocampal neurogenesis at the early stages of Alzheimer’s disease (AD) is believed to support early cognitive decline. Converging studies sustain the idea that vitamin D might be linked to the pathophysiology of AD and to hippocampal neurogenesis. Nothing being known about the effects of vitamin D on hippocampal neurogenesis in AD, we assessed them in a mouse model of AD. In a previous study, we observed that dietary vitamin D supplementation in female AD-like mice reduced cognitive decline only when delivered during the symptomatic phase. With these data in hand, we wondered whether the consequences of vitamin D administration on hippocampal neurogenesis are stage-dependent. Male wild-type and transgenic AD-like mice (5XFAD model) were fed with a diet containing either no vitamin D (0VD) or a normal dose of vitamin D (NVD) or a high dose of vitamin D (HVD), from month 1 to month 6 (preventive arm) or from month 4 to month 9 (curative arm). Working memory was assessed using the Y-maze, while amyloid burden, astrocytosis, and neurogenesis were quantified using immunohistochemistry. In parallel, the effects of vitamin D on proliferation and differentiation were assayed on primary cultures of murine neural progenitor cells. Improved working memory and neurogenesis were observed when high vitamin D supplementation was administered during the early phases of the disease, while a normal dose of vitamin D increased neurogenesis during the late phases. Conversely, an early hypovitaminosis D increased the number of amyloid plaques in AD mice while a late hypovitaminosis D impaired neurogenesis in AD and WT mice. The observed in vivo vitamin D-associated increased neurogenesis was partially substantiated by an augmented in vitro proliferation but not an increased differentiation of neural progenitors into neurons. Finally, a sexual dimorphism was observed. Vitamin D supplementation improved the working memory of males and females, when delivered during the pre-symptomatic and symptomatic phases, respectively. Our study establishes that (i) neurogenesis is improved by vitamin D in a male mouse model of AD, in a time-dependent manner, and (ii) cognition is enhanced in a gender-associated way. Additional pre-clinical studies are required to further understand the gender- and time-specific mechanisms of action of vitamin D in AD. This may lead to an adaptation of vitamin D supplementation in relation to patient’s gender and age as well as to the stage of the disease.

Peptide nanosponges designed for rapid uptake by leukocytes and neural stem cells.

The structure of novel binary nanosponges consisting of (cholesterol-(K/D)nDEVDGC)3-trimaleimide units possessing a trigonal maleimide linker, to which either lysine (K)20 or aspartic acid (D)20 are tethered, has been elucidated by means of TEM. A high degree of agreement between these findings and structure predictions through explicit solvent and then coarse-grained molecular dynamics (MD) simulations has been found. Based on the nanosponges' structure and dynamics, caspase-6 mediated release of the model drug 5(6)-carboxyfluorescein has been demonstrated. Furthermore, the binary (DK20) nanosponges have been found to be virtually non-toxic in cultures of neural progenitor cells. It is of a special importance for the future development of cell-based therapies that DK20 nanosponges were taken up efficiently by leucocytes (WBC) in peripheral blood within 3 h of exposure. The percentage of live cells among the WBC was not significantly decreased by the DK20 nanosponges. In contrast to stem cell or leucocyte cell cultures, which have to be matched to the patient, autologous cells are optimal for cell-mediated therapy. Therefore, the nanosponges hold great promise for effective cell-based tumor targeting.

Derepression of the Iroquois Homeodomain Transcription Factor Gene IRX3 Confers Differentiation Block in Acute Leukemia

SummaryThe Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in ∼30% of patients with acute myeloid leukemia (AML), ∼50% with T-acute lymphoblastic leukemia, and ∼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.

Transcriptional profile of in vitro expanded human epidermal progenitor cells for the treatment of non-healing wounds

BackgroundEpidermal progenitor cells (EPCs) have been under extensive investigation due to their increasing potential of application in medicine and biotechnology. Cultured human EPCs are used in the treatment of chronic wounds and have recently became a target for gene therapy and toxicological studies. One of the challenges in EPCs culture is to provide a high number of undifferentiated, progenitor cells displaying high viability and significant biological activity. ObjectivesThe goal of this study was to characterize the in vitro cultured progenitor cells and to assess whether the cells with the progenitor phenotype are able to enhance wound healing. Additionally, we aimed to establish the complete procedure of the culture, analysis and clinical application of epidermal progenitor cells. MethodsIn this study we present a method of cell isolation and culture followed by a technique of transplantation of the cultured cells onto the wound bed. The applied isolation technique involves two enzymatic steps (dispase, trypsin) and it is characterized by a high yield of cells. The obtained cells were cultured in vitro up to the second passage in serum-free and xeno-free keratinocytes-dedicated medium. Key stem cell markers were determined with means of flow cytometry and quantitative real-time PCR. ResultsThe in vitro expanded cells displayed high proliferative activity without features of neither apoptosis nor necrosis. The flow cytometry and transcriptomic analyses showed enhanced expression of stem cell markers (i.e. proteins: ΔNp63, CD29, CD49f and BNC1, CDKN1A transcripts) in the expanded cells. In the presented compassionate use study, cultured autologous cells from an oncological patient were suspended in fibrin sealant and transplanted directly to a non-healing wound, resulting in wound closure within 2 months. ConclusionThe cells cultured in serum-free media display epidermal stem cells features and a potential to stimulate wound healing. This promising procedure of isolation, culture and application warrants further clinical trials in the treatment of chronic wounds.

Oligodendrocyte Progenitor Cell Cultures: A Model to Screen Neurotrophic Compounds for Myelin Repair.

The protocol presented in this chapter covers the application of rat cortical oligodendrocyte progenitor cells cultured under conditions of differentiation for the evaluation of agents with potential trophic activity, that is, which are capable of promoting maturation under such conditions. As an example we have chosen a co-ultramicronized N-palmitoylethanolamine/luteolin composite, a formulation described in the literature as possessing anti-inflammatory, neuroprotective, and neuroregenerative actions.

Down Syndrome iPSC-Derived Astrocytes Impair Neuronal Synaptogenesis and the mTOR Pathway In Vitro.

Several methods have been used to study the neuropathogenesis of Down syndrome (DS), such as mouse aneuploidies, post mortem human brains, and in vitro cell culture of neural progenitor cells. More recently, induced pluripotent stem cell (iPSC) technology has offered new approaches in investigation, providing a valuable tool for studying specific cell types affected by DS, especially neurons and astrocytes. Here, we investigated the role of astrocytes in DS developmental disease and the impact of the astrocyte secretome in neuron mTOR signaling and synapse formation using iPSC derived from DS and wild-type (WT) subjects. We demonstrated for the first time that DS neurons derived from hiPSC recapitulate the hyperactivation of the Akt/mTOR axis observed in DS brains and that DS astrocytes may play a key role in this dysfunction. Our results bear out that 21 trisomy in astrocytes contributes to neuronal abnormalities in addition to cell autonomous dysfunctions caused by 21 trisomy in neurons. Further research in this direction will likely yield additional insights, thereby improving our understanding of DS and potentially facilitating the development of new therapeutic approaches.

Induction of deubiquitinating enzyme USP50 during erythropoiesis and its potential role in the regulation of Ku70 stability

Anemia is a very common blood disorder that affects the lives of billions of people worldwide. Anemia is caused by the loss of blood, increased destruction of red blood cells...

Maintenance of neural progenitor cell stemness in 3D hydrogels requires matrix remodelling.

Neural progenitor cell (NPC) culture within 3D hydrogels is an attractive strategy for expanding a therapeutically-relevant number of stem cells. However, relatively little is known about how 3D material properties such as stiffness and degradability affect the maintenance of NPC stemness in the absence of differentiation factors. Over a physiologically-relevant range of stiffness from ~0.5–50 kPa, stemness maintenance did not correlate with initial hydrogel stiffness. In contrast, hydrogel degradation was both correlated with, and necessary for, maintenance of NPC stemness. This requirement for degradation was independent of cytoskeletal tension generation and presentation of engineered adhesive ligands, instead relying on matrix remodeling to facilitate cadherin-mediated cell-cell contact and promote β-catenin signaling. In two additional hydrogel systems, permitting NPC-mediated matrix remodeling proved to be a generalizable strategy for stemness maintenance in 3D. Our findings have identified matrix remodeling, in the absence of cytoskeletal tension generation, as a previously unknown strategy to maintain stemness in 3D.

Comparative Analysis of Mesenchymal Progenitor Cells from Dental Pulp and Cord Tissue and their Potentiality Towards Trans-Differentiation

Objective: The objective of our study was to successfully isolate progenitor cells from dental pulp and umbilical tissue and perform a comparative investigation of their potential transdifferentiation into osteo- and neuronal-like cells. Methods: Progenitor cells were harvested from dental pulp tissue as well as cord tissue and cultured through explant culture method over the span of 4 weeks. Image-based cytometric analysis was done to determine the cell viability along with phenotypic analysis to validate the occurrence of stem cell surface markers such as CD13, CD29, CD31, CD34, CD45, CD73, CD90, CD105, HLA-DR, and HLA-ABC. After culturing mesenchymal progenitor cells, osteogenic and neurogenic differentiation potential of both tissue sources was studied. The cells were seeded in two different surfaces tissue culture treated dishes and titanium sheets and cultured along with osteogenic differentiation medium (for 28 days) and neurogenic differentiation medium (for 5 days). The osteogenic potential of the progenitor cells were checked with the detection of calcium deposits by Vonn Kossa staining and PCR studies were done to confirm the presence of osteogenic genes like BMP2, HDAC1, HNF1A. The neurogenic potential of the progenitors were phenotypically determined by the observation of neuronal cells in the culture medium. Post differentiation PCR studies were done to confirm the presence of neuronal genes like NESTIN, AGRIN, MAG, DAPDH, NF-M. Findings: Progenitor cells extracted from cord tissue and dental pulp were positive for markers such as CD13, CD29, CD73, CD90, and CD105 and were found negative for markers such CD31, CD34, CD45, and HLA-DR. Progenitors obtained from dental pulp tissue showed a higher expression of cell surface markers indicating a stronger mesenchymal lineage. After culturing progenitor cells in osteogenic differentiation specific medium, these cells were successfully differentiated into cells of osteogenic lineage. Within 28 days of culture calcium deposits were detected by Von kossa staining. The differentiated cells were also found positive for osteogenic markers such as BMP2, HDAC1, HNF1A.In neural differentiation, post day 5 of culture neurospheres of varying sizes were observed floating in the culture medium. The fraction of the cells differentiated into osteogenic and neurogenic lineages were higher in progenitor cells derived from dental pulp in comparison with umbilical cord tissue. The higher potentialityof progenitor cells derived from dental pulp for neurogenic trans-differentiation could be explained by the fact that human adult dental pulp stem cells residing within the perivascular niche are thought to originate from the migrating cranial neural crest cells. There was no difference observed in the osteogenic and neurogenic differentiation capabilities of mesenchymal cells when plated in a plastic dish or titanium surface. Improvement: In-depth studies needs to be carried out on progenitor cells from dental pulp tissues in order to enhance the clinical efficacy of stem cells based therapies.

Standardization of a protocol to irradiate cell cultures with low level laser

Introduction: It is impossible to apply orthodontic therapeutic laser during the orthodontic treatment and to select different times of exposure, frequency or intensity for every single cell. The whole area gets irradiated in seconds according to the laser type . This could have a proliferation effect in some cells and a harmful effect in others cells. So, it´s very important to asses the effects of a laser application on the cells involved in dental movement, to verify the effect and reaction after laser therapy.&#x0D; Objective: To create and standarized a clinical protocol to irradiate culture cells involved in dental movement (Human fibroblasts, osteoblasts and pre-osteoclasts in order to understand the basics of it´s clinical effect.&#x0D; Materials and methods: Gingival and periodontal fibroblasts, normal human osteoblasts (NHOst) and human osteoclast progenitor cells in culture were irradiated using a low energy level laser (As-GaAl), operated at 36.73mW, 832.79nm nm wavelength 3.75 J/cm2 and 32.40 seconds per well.&#x0D; Conclusions: The cytotoxicity tests showed that irradiation is safe protocol for the four groups of cell cultures.

Jak kinase 3 signaling in microgliogenesis from the spinal nestin+ progenitors in both development and response to injury.

During spinal cord development, endogenous progenitors expressing nestin can migrate into the target and differentiate into neurons and other glial cells. Microglial cells can also be derived from nestin+ progenitor cells, even in the adult brain. Knockdown of Jak kinase 3 (Jak3) signaling can increase neurogenesis with longer neurite outgrowth in cortical progenitor cells. This study investigated the effect of Jak3 signaling on differentiation from nestin+ progenitor cells using E13.5 spinal progenitor cell cultures. In growth factors-enriched culture, developing neurons could not survive after several days and also a significant proportion of nestin-expressing cells transformed into ameboid Iba1+ microglial cells, which increased exponentially after 5 days. This microgliogenesis was predominantly regulated by Jak3 signaling, which was confirmed by transcription factors responsible for microgliogenesis, and microglial migration and phagocytosis, such as Pu.1, Irf8, and Runx1. Jak3 inhibition also significantly increased the Tuj1+ growing neurites with little microglial activation. These results indicated that neuronal and microglial cell differentiation was regulated primarily by Jak3 signaling and the developing neurons and neurite outgrowth might also be regulated by Jak3-dependent microglial activity.