Donor variability alters differentiation and mechanical cohesion of tissue-engineered constructs with human endothelial/MSC co-culture

To move towards clinical applications, tissue engineering (TE) should be validated with human primary cells and offer easy connection to the native vascularisation. Based on a sheet-like bone substitute developed previously, we investigated a mesenchymal stem cells/endothelial cells (MSCs/ECs) coculture to enhance pre-vascularisation. Using MSCs from six independent donors whose differentiation potential was assessed towards two lineages, we focused on donor variability and cell crosstalk regarding bone differentiation. Coculture was performed on calcium phosphate granules in a specific chamber during 1 month. MSCs were seeded first then ECs were added after 2 weeks, with respective monocultures as control groups. Cell viability and organisation (fluorescence, electronic microscopy), differentiation (ALP staining/activity, RT-qPCR) and mechanical cohesion were analysed. Adaptation of the protocol to coculture was validated (high cell viability and proliferation). Activity and differentiation showed strong trends towards synergistic effects between cell types. MSCs reached early mineralisation stage of maturation. The delayed addition of ECs allowed for their attachment on developed MSCs’ matrix. The main impact of donor variability could be here the lack of cell proliferation potential with some donors, leading to low differentiation and mechanical cohesion and therefore absence of sheet-like shape successfully obtained with others. We suggest therefore adapting protocols to cell proliferation potentials from one batch of cells to the other in a patient-specific approach.

Download Full-text

MRTF-A and YAP exert dual control in GPCR and RhoA-mediated transcriptional regulation and cell proliferation

Molecular and Cellular Biology ◽

10.1128/mcb.00772-15 ◽

2015 ◽

pp. MCB.00772-15 ◽

Cited By ~ 17

Author(s):

Olivia Yu ◽

Shigeki Miyamoto ◽

Joan Heller Brown

Keyword(s):

Gene Expression ◽

Cell Proliferation ◽

Synergistic Effects ◽

Cell Types ◽

Luciferase Expression ◽

Regulated Gene Expression ◽

Potential Interactions ◽

G Protein Coupled ◽

Regulatory Properties ◽

Chip Analysis

The ability of a subset of G-protein coupled receptors (GPCRs) to activate RhoA endows them with unique growth regulatory properties. Two transcriptional pathways are activated through GPCRs and RhoA, one utilizing SRF and its transcriptional co-activator MRTF-A, the other, TEAD and its transcriptional co-activator YAP. These pathways have not been compared for their relative importance and potential interactions in RhoA target gene expression. GPCRs for thrombin and S1P on human glioblastoma cells robustly couple to RhoA and induce the matricelluar protein CCN1. Knockdown of either MRTF-A or YAP abrogates S1P stimulated CCN1 expression, demonstrating that both co-activators are required. MRTF-A and YAP are also both required for induction of other S1P regulated genes in various cell types and for S1P stimulated glioblastoma cell proliferation. Interactions between MRTF-A and YAP are indicated by their synergistic effects on SRE.L and TEAD-luciferase expression. Moreover, MRTF-A and YAP associate in co-IPs from S1P stimulated cells. ChIP analysis of the CCN1 promoter demonstrates that S1P increases co-activator binding at their canonical transcription factor sequences. Unexpectedly, SIP also enhances MRTF-A binding at TEA sites. Our findings reveal that GPCR and RhoA-regulated gene expression requires dual input and integration of two distinct transcriptional pathways.

Download Full-text

Uncovering the Diversification of Tissue Engineering on the Emergent Areas of Stem Cells, Nanotechnology and Biomaterials

Current Stem Cell Research & Therapy ◽

10.2174/1574888x15666200103124821 ◽

2020 ◽

Vol 15 (3) ◽

pp. 187-201 ◽

Cited By ~ 1

Author(s):

Sunil K. Dubey ◽

Amit Alexander ◽

Munnangi Sivaram ◽

Mukta Agrawal ◽

Gautam Singhvi ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Immune System ◽

Clinical Application ◽

Cell Types ◽

Patient Specific ◽

Potential Strategy ◽

Induced Pluripotent ◽

Treat All ◽

The Impact

Damaged or disabled tissue is life-threatening due to the lack of proper treatment. Many conventional transplantation methods like autograft, iso-graft and allograft are in existence for ages, but they are not sufficient to treat all types of tissue or organ damages. Stem cells, with their unique capabilities like self-renewal and differentiate into various cell types, can be a potential strategy for tissue regeneration. However, the challenges like reproducibility, uncontrolled propagation and differentiation, isolation of specific kinds of cell and tumorigenic nature made these stem cells away from clinical application. Today, various types of stem cells like embryonic, fetal or gestational tissue, mesenchymal and induced-pluripotent stem cells are under investigation for their clinical application. Tissue engineering helps in configuring the stem cells to develop into a desired viable tissue, to use them clinically as a substitute for the conventional method. The use of stem cell-derived Extracellular Vesicles (EVs) is being studied to replace the stem cells, which decreases the immunological complications associated with the direct administration of stem cells. Tissue engineering also investigates various biomaterials to use clinically, either to replace the bones or as a scaffold to support the growth of stemcells/ tissue. Depending upon the need, there are various biomaterials like bio-ceramics, natural and synthetic biodegradable polymers to support replacement or regeneration of tissue. Like the other fields of science, tissue engineering is also incorporating the nanotechnology to develop nano-scaffolds to provide and support the growth of stem cells with an environment mimicking the Extracellular matrix (ECM) of the desired tissue. Tissue engineering is also used in the modulation of the immune system by using patient-specific Mesenchymal Stem Cells (MSCs) and by modifying the physical features of scaffolds that may provoke the immune system. This review describes the use of various stem cells, biomaterials and the impact of nanotechnology in regenerative medicine.

Download Full-text

Extracellular-Vesicle-Based Coatings Enhance Bioactivity of Titanium Implants—SurfEV

Nanomaterials ◽

10.3390/nano11061445 ◽

2021 ◽

Vol 11 (6) ◽

pp. 1445

Author(s):

Taisa Nogueira Pansani ◽

Thanh Huyen Phan ◽

Qingyu Lei ◽

Alexey Kondyurin ◽

Bill Kalionis ◽

...

Keyword(s):

Cell Proliferation ◽

Titanium Surface ◽

Wound Closure ◽

Cell Types ◽

Extracellular Vesicle ◽

Tissue Growth ◽

Titanium Implants ◽

The Body ◽

High Concentrations ◽

And Migration

Extracellular vesicles (EVs) are nanoparticles released by cells that contain a multitude of biomolecules, which act synergistically to signal multiple cell types. EVs are ideal candidates for promoting tissue growth and regeneration. The tissue regenerative potential of EVs raises the tantalizing possibility that immobilizing EVs on implant surfaces could potentially generate highly bioactive and cell-instructive surfaces that would enhance implant integration into the body. Such surfaces could address a critical limitation of current implants, which do not promote bone tissue formation or bond bone. Here, we developed bioactive titanium surface coatings (SurfEV) using two types of EVs: secreted by decidual mesenchymal stem cells (DEVs) and isolated from fermented papaya fluid (PEVs). For each EV type, we determined the size, morphology, and molecular composition. High concentrations of DEVs enhanced cell proliferation, wound closure, and migration distance of osteoblasts. In contrast, the cell proliferation and wound closure decreased with increasing concentration of PEVs. DEVs enhanced Ca/P deposition on the titanium surface, which suggests improvement in bone bonding ability of the implant (i.e., osteointegration). EVs also increased production of Ca and P by osteoblasts and promoted the deposition of mineral phase, which suggests EVs play key roles in cell mineralization. We also found that DEVs stimulated the secretion of secondary EVs observed by the presence of protruding structures on the cell membrane. We concluded that, by functionalizing implant surfaces with specialized EVs, we will be able to enhance implant osteointegration by improving hydroxyapatite formation directly at the surface and potentially circumvent aseptic loosening of implants.

Download Full-text

Combined Application of Pan-AKT Inhibitor MK-2206 and BCL-2 Antagonist Venetoclax in B-Cell Precursor Acute Lymphoblastic Leukemia

International Journal of Molecular Sciences ◽

10.3390/ijms22052771 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2771

Author(s):

Anna Richter ◽

Elisabeth Fischer ◽

Clemens Holz ◽

Julia Schulze ◽

Sandra Lange ◽

...

Keyword(s):

Cell Proliferation ◽

Tumor Cell ◽

Cell Lines ◽

Morphological Changes ◽

Synergistic Effects ◽

Lymphoblastic Leukemia ◽

Tumor Cell Proliferation ◽

Akt Inhibitor ◽

Akt Phosphorylation ◽

Combined Application

Aberrant PI3K/AKT signaling is a hallmark of acute B-lymphoblastic leukemia (B-ALL) resulting in increased tumor cell proliferation and apoptosis deficiency. While previous AKT inhibitors struggled with selectivity, MK-2206 promises meticulous pan-AKT targeting with proven anti-tumor activity. We herein, characterize the effect of MK-2206 on B-ALL cell lines and primary samples and investigate potential synergistic effects with BCL-2 inhibitor venetoclax to overcome limitations in apoptosis induction. MK-2206 incubation reduced AKT phosphorylation and influenced downstream signaling activity. Interestingly, after MK-2206 mono application tumor cell proliferation and metabolic activity were diminished significantly independently of basal AKT phosphorylation. Morphological changes but no induction of apoptosis was detected in the observed cell lines. In contrast, primary samples cultivated in a protective microenvironment showed a decrease in vital cells. Combined MK-2206 and venetoclax incubation resulted in partially synergistic anti-proliferative effects independently of application sequence in SEM and RS4;11 cell lines. Venetoclax-mediated apoptosis was not intensified by addition of MK-2206. Functional assessment of BCL-2 inhibition via Bax translocation assay revealed slightly increased pro-apoptotic signaling after combined MK-2206 and venetoclax incubation. In summary, we demonstrate that the pan-AKT inhibitor MK-2206 potently blocks B-ALL cell proliferation and for the first time characterize the synergistic effect of combined MK-2206 and venetoclax treatment in B-ALL.

Download Full-text

Generation and Evaluation of Isogenic iPSC as a Source of Cell Replacement Therapies in Patients with Kearns Sayre Syndrome

Cells ◽

10.3390/cells10030568 ◽

2021 ◽

Vol 10 (3) ◽

pp. 568

Author(s):

Glen Lester Sequiera ◽

Abhay Srivastava ◽

Keshav Narayan Alagarsamy ◽

Cheryl Rockman-Greenberg ◽

Sanjiv Dhingra

Keyword(s):

Mononuclear Cells ◽

Therapeutic Option ◽

Reactive Oxygen Species Generation ◽

Energy Deficit ◽

Patient Specific ◽

Multisystem Disorder ◽

Differentiation Potential ◽

Cell Replacement ◽

Mtdna Mutations ◽

Kearns Sayre Syndrome

Kearns Sayre syndrome (KSS) is mitochondrial multisystem disorder with no proven effective treatment. The underlying cause for multisystem involvement is the energy deficit resulting from the load of mutant mitochondrial DNA (mtDNA), which manifests as loss of cells and tissue dysfunction. Therefore, functional organ or cellular replacement provides a promising avenue as a therapeutic option. Patient-specific induced pluripotent stem cells (iPSC) have become a handy tool to create personalized cell -based therapies. iPSC are capable of self-renewal, differentiation into all types of body cells including cardiomyocytes (CM) and neural progenitor cells (NPC). In KSS patients, mutations in mtDNA are largely found in the muscle tissue and are predominantly absent in the blood cells. Therefore, we conceptualized that peripheral blood mononuclear cells (PBMNC) from KSS patients can be reprogrammed to generate mutation free, patient specific iPSC lines that can be used as isogenic source of cell replacement therapies to treat affected organs. In the current study we generated iPSC lines from two female patients with clinical diagnosis of classic KSS. Our data demonstrate that iPSC from these KSS patients showed normal differentiation potential toward CM, NPC and fibroblasts without any mtDNA deletions over passages. Next, we also found that functional studies including ATP production, reactive oxygen species generation, lactate accumulation and mitochondrial membrane potential in iPSC, CM, NPC and fibroblasts of these KSS patients were not different from respective cells from healthy controls. PBMNCs from these KSS patients in the current study did not reproduce mtDNA mutations which were present in muscle biopsies. Furthermore, we demonstrate for the first time that this phenomenon provides opportunities to create isogenic mutation free iPSC with absent or very low level of expression of mtDNA deletion which can be banked for future cell replacement therapies in these patients as the disease progresses.

Download Full-text

Bone marrow stromal cells from MDS and AML patients show increased adipogenic potential with reduced Delta-like-1 expression

Scientific Reports ◽

10.1038/s41598-021-85122-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Marie-Theresa Weickert ◽

Judith S. Hecker ◽

Michèle C. Buck ◽

Christina Schreck ◽

Jennifer Rivière ◽

...

Keyword(s):

Bone Marrow ◽

Cell Fate ◽

Stromal Cells ◽

Adipogenic Differentiation ◽

Cell Types ◽

Bone Marrow Niche ◽

Differentiation Potential ◽

Hematopoietic Stem ◽

Bm Niche

AbstractMyelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are clonal hematopoietic stem cell disorders with a poor prognosis, especially for elderly patients. Increasing evidence suggests that alterations in the non-hematopoietic microenvironment (bone marrow niche) can contribute to or initiate malignant transformation and promote disease progression. One of the key components of the bone marrow (BM) niche are BM stromal cells (BMSC) that give rise to osteoblasts and adipocytes. It has been shown that the balance between these two cell types plays an important role in the regulation of hematopoiesis. However, data on the number of BMSC and the regulation of their differentiation balance in the context of hematopoietic malignancies is scarce. We established a stringent flow cytometric protocol for the prospective isolation of a CD73+ CD105+ CD271+ BMSC subpopulation from uncultivated cryopreserved BM of MDS and AML patients as well as age-matched healthy donors. BMSC from MDS and AML patients showed a strongly reduced frequency of CFU-F (colony forming unit-fibroblast). Moreover, we found an altered phenotype and reduced replating efficiency upon passaging of BMSC from MDS and AML samples. Expression analysis of genes involved in adipo- and osteogenic differentiation as well as Wnt- and Notch-signalling pathways showed significantly reduced levels of DLK1, an early adipogenic cell fate inhibitor in MDS and AML BMSC. Matching this observation, functional analysis showed significantly increased in vitro adipogenic differentiation potential in BMSC from MDS and AML patients. Overall, our data show BMSC with a reduced CFU-F capacity, and an altered molecular and functional profile from MDS and AML patients in culture, indicating an increased adipogenic lineage potential that is likely to provide a disease-promoting microenvironment.

Download Full-text

Optimization of Spheroid Colony Culture and Cryopreservation of Nucleus Pulposus Cells for the Development of Intervertebral Disc Regenerative Therapeutics

Applied Sciences ◽

10.3390/app11083309 ◽

2021 ◽

Vol 11 (8) ◽

pp. 3309

Author(s):

Kosuke Sako ◽

Daisuke Sakai ◽

Yoshihiko Nakamura ◽

Erika Matsushita ◽

Jordy Schol ◽

...

Keyword(s):

Cell Proliferation ◽

Cell Viability ◽

Nucleus Pulposus ◽

Mass Culture ◽

Formation Rate ◽

Proliferation Rate ◽

Cell Phenotype ◽

Mrna Levels ◽

Spheroid Formation ◽

Positive Nucleus

After the discovery of functionally superior Tie2-positive nucleus pulposus (NP) progenitor cells, new methods were needed to enable mass culture and cryopreservation to maintain these cells in an undifferentiated state with high cell yield. We used six types of EZSPHERE® dishes, which support spheroid-forming colony culture, and examined NP cell spheroid-formation ability, number, proliferation, and mRNA expression of ACAN, COL1A2, COL2A1, and ANGPT1. Six different types of cryopreservation solutions were examined for potential use in clinical cryopreservation by comparing the effects of exposure time during cryopreservation on cell viability, Tie2-positivity, and cell proliferation rates. The spheroid formation rate was 45.1% and the cell proliferation rate was 7.75 times using EZSPHERE® dishes. The mRNA levels for COL2A1 and ANGPT1 were also high. In cryopreservation, CryoStor10 (CS10) produced ≥90% cell viability and a high proliferation rate after thawing. CS10 had a high Tie2-positive rate of 12.6% after culturing for 5 days after thawing. These results suggest that EZSPHERE enabled colony formation in cell culture without the use of hydrogel products and that CS10 is the best cryopreservation medium for retaining the NP progenitor cell phenotype and viability. Together, these data provide useful information of NP cell-based therapeutics to the clinic.

Download Full-text

Resveratrol Enhances Inhibition Effects of Cisplatin on Cell Migration and Invasion and Tumor Growth in Breast Cancer MDA-MB-231 Cell Models In Vivo and In Vitro

Molecules ◽

10.3390/molecules26082204 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2204

Author(s):

Meng-Die Yang ◽

Yang Sun ◽

Wen-Jun Zhou ◽

Xiao-Zheng Xie ◽

Qian-Mei Zhou ◽

...

Keyword(s):

Breast Cancer ◽

Cell Migration ◽

Tumor Growth ◽

Cell Viability ◽

Synergistic Effects ◽

Migration And Invasion ◽

Cell Migration And Invasion ◽

Tgf Β1

Triple-negative breast cancer (TNBC) is a refractory type of breast cancer that does not yet have clinically effective drugs. The aim of this study is to investigate the synergistic effects and mechanisms of resveratrol combined with cisplatin on human breast cancer MDA-MB-231 (MDA231) cell viability, migration, and invasion in vivo and in vitro. In vitro, MTS assays showed that resveratrol combined with cisplatin inhibits cell viability as a concentration-dependent manner, and produced synergistic effects (CI < 1). Transwell assay showed that the combined treatment inhibits TGF-β1-induced cell migration and invasion. Immunofluorescence assays confirmed that resveratrol upregulated E-cadherin expression and downregulated vimentin expression. Western blot assay demonstrated that resveratrol combined with cisplatin significantly reduced the expression of fibronectin, vimentin, P-AKT, P-PI3K, P-JNK, P-ERK, Sma2, and Smad3 induced by TGF-β1 (p < 0.05), and increased the expression of E-cadherin (p < 0.05), respectively. In vivo, resveratrol enhanced tumor growth inhibition and reduced body weight loss and kidney function impairment by cisplatin in MDA231 xenografts, and significantly reduced the expressions of P-AKT, P-PI3K, Smad2, Smad3, P-JNK, P-ERK, and NF-κB in tumor tissues (p < 0.05). These results indicated that resveratrol combined with cisplatin inhibits the viability of breast cancer MDA231 cells synergistically, and inhibits MDA231 cells invasion and migration through Epithelial-mesenchymal transition (EMT) approach, and resveratrol enhanced anti-tumor effect and reduced side of cisplatin in MDA231 xenografts. The mechanism may be involved in the regulations of PI3K/AKT, JNK, ERK and NF-κB expressions.

Download Full-text

The Olive Leaves Extract Has Anti-Tumor Effects against Neuroblastoma through Inhibition of Cell Proliferation and Induction of Apoptosis

Nutrients ◽

10.3390/nu13072178 ◽

2021 ◽

Vol 13 (7) ◽

pp. 2178

Author(s):

Fabio Morandi ◽

Veronica Bensa ◽

Enzo Calarco ◽

Fabio Pastorino ◽

Patrizia Perri ◽

...

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Viability ◽

Cell Cycle Progression ◽

Treatment Strategies ◽

Annexin V ◽

Dependent Manner ◽

Induction Of Apoptosis ◽

Dose Dependent

Neuroblastoma (NB) is the most common extra-cranial solid tumor of pediatric age. The prognosis for high-risk NB patients remains poor, and new treatment strategies are desirable. The olive leaf extract (OLE) is constituted by phenolic compounds, whose health beneficial effects were reported. Here, the anti-tumor effects of OLE were investigated in vitro on a panel of NB cell lines in terms of (i) reduction of cell viability; (ii) inhibition of cell proliferation through cell cycle arrest; (iii) induction of apoptosis; and (iv) inhibition of cell migration. Furthermore, cytotoxicity experiments, by combining OLE with the chemotherapeutic topotecan, were also performed. OLE reduced the cell viability of NB cells in a time- and dose-dependent manner in 2D and 3D models. NB cells exposed to OLE underwent inhibition of cell proliferation, which was characterized by an arrest of the cell cycle progression in G0/G1 phase and by the accumulation of cells in the sub-G0 phase, which is peculiar of apoptotic death. This was confirmed by a dose-dependent increase of Annexin V+ cells (peculiar of apoptosis) and upregulation of caspases 3 and 7 protein levels. Moreover, OLE inhibited the migration of NB cells. Finally, the anti-tumor efficacy of the chemotherapeutic topotecan, in terms of cell viability reduction, was greatly enhanced by its combination with OLE. In conclusion, OLE has anti-tumor activity against NB by inhibiting cell proliferation and migration and by inducing apoptosis.

Download Full-text

NEAT1/miR-140-3p/MAPK1 mediates the viability and survival of coronary endothelial cells and affects coronary atherosclerotic heart disease

Acta Biochimica et Biophysica Sinica ◽

10.1093/abbs/gmaa087 ◽

2020 ◽

Vol 52 (9) ◽

pp. 967-974

Author(s):

Hui Zhang ◽

Ningning Ji ◽

Xinyan Gong ◽

Shimao Ni ◽

Yu Wang

Keyword(s):

Cell Proliferation ◽

Endothelial Cells ◽

Heart Disease ◽

Cell Viability ◽

Mitogen Activated Protein Kinase ◽

Expression Level ◽

Luciferase Reporter ◽

Coronary Endothelial Cells ◽

Induced Apoptosis ◽

Lncrna Neat1

Abstract Studies have shown that long non-coding RNAs (lncRNA) play critical roles in coronary atherosclerotic heart disease (CAD). However, the function of lncRNA nuclear enriched abundant transcript 1 (NEAT1) in CAD is unclear. In this study, we aimed to investigate the functions of lncRNA NEAT1 in CAD. RT-PCR and western blot analysis were carried out to examine the expressions of related RNAs. Colony formation assay, cell proliferation assay, apoptosis assay, and dual-luciferase reporter assay were conducted to investigate the abilities of colony migration, cell proliferation, apoptosis, and targeting. The results showed that NEAT1 was up-regulated in CAD blood samples and in human coronary endothelial cells (HCAECs). Transfection of pcNEAT1 significantly inhibited the survival rate of HCAECs and induced apoptosis of HCAECs. MiR-140-3p was down-regulated in HCAECs. NEAT1 directly targeted miR-140-3p, and the expression of miR-140-3p was inversely correlated with the expression of NEAT1 in CAD patients. In addition, co-transfection of NEAT1 with miR-140-3p mimic reversed the effect of pcNEAT1 on cell viability and apoptosis. mitogen-activated protein kinase 1 (MAPK1) was proved to be a target gene of miR-140-3p, and the miR-140-3p mimic was shown to reduce the expression of MAPK1 in HCAECs. pcNEAT1 significantly increased the expression level of MAPK1, while shNEAT1 significantly reduced the expression level of MAPK1. Our results revealed that lncRNA NEAT1 increased cell viability and inhibited CAD cell apoptosis possibly by activating the miR-140-3p/MAPK1 pathway, and lncRNA NEAT1 might serve as a potential therapeutic target for CAD.

Download Full-text