scholarly journals Serum-free culture system for spontaneous human mesenchymal stem cell spheroids formation

2019 ◽  
Author(s):  
Guoyi Dong ◽  
Shengpeng Wang ◽  
Yuping Ge ◽  
Qiuting Deng ◽  
Qi Cao ◽  
...  
Keyword(s):  
Clinical Research ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cell ◽  
Culture Methods ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement

AbstractHuman mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids, but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stems medium (MiPS), adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were up-regulated in spheroids at day 6, and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were re-plated in normal FBS medium, cells formed a typical spindle-shaped morphology, and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

scholarly journals Serum-Free Culture System for Spontaneous Human Mesenchymal Stem Cell Spheroid Formation

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Guoyi Dong ◽  
Shengpeng Wang ◽  
Yuping Ge ◽  
Qiuting Deng ◽  
Qi Cao ◽  
...  
Keyword(s):  
Clinical Research ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Human Mesenchymal Stem Cells ◽  
Human Mesenchymal Stem Cell ◽  
Spheroid Formation ◽  
Culture Methods ◽  
Serum Replacement ◽  
Knockout Serum Replacement

Human mesenchymal stem cells (hMSCs) are widely used in clinical research because of their multipotential, immunomodulatory, and reparative properties. Previous studies determined that hMSC spheroids from a three-dimensional (3D) culture possess higher therapeutic efficacy than conventional hMSCs from a monolayer (2D) culture. To date, various 3D culture methods have been developed to form hMSC spheroids but most of them used culture medium containing fetal bovine serum (FBS), which is not suitable for further clinical use. Here, we demonstrate that dissociated single MSCs seeded in induced pluripotent stem medium (MiPS) adhere loosely to the dish and spontaneously migrate to form spheroids during day 3 to day 6. Through component deletion screening and complementation experiments, the knockout serum replacement (KSR) was identified as necessary and sufficient for hMSC spheroid formation. Transcriptome analysis showed that the overall expression profiles were highly similar between 2D culture with FBS and KSR-derived spheroids. Interestingly, genes related to inflammatory response, immune response, and angiogenesis were upregulated in spheroids at day 6 and qPCR results further validated the increased expression level of related genes, including STC1, CCL7, HGF, IL24, and TGFB3. When spheroids were replated in normal FBS medium, cells formed a typical spindle-shaped morphology and FACS results showed that the recovered cells retained MSC-specific surface markers, such as CD73, CD90, and CD105. In summary, we developed a practical and convenient method to generate hMSC spheroids for clinical research and therapy.

scholarly journals Neurosphere Based Differentiation of Human iPSC Improves Astrocyte Differentiation

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Shuling Zhou ◽  
Karolina Szczesna ◽  
Anna Ochalek ◽  
Julianna Kobolák ◽  
Eszter Varga ◽  
...  
Keyword(s):  
Neural Progenitor Cells ◽  
Three Dimensional ◽  
3D Culture ◽  
Neural Progenitor Cell ◽  
Neural Progenitor ◽  
Culture Methods ◽  
Differentiation Potential ◽  
Differentiated Cells ◽  
Astrocyte Differentiation ◽  
Induced Pluripotent

Neural progenitor cells (NPCs) derived from human induced pluripotent stem cells (iPSCs) are traditionally maintained and proliferated utilizing two-dimensional (2D) adherent monolayer culture systems. However, NPCs cultured using this system hardly reflect the intrinsic spatial development of brain tissue. In this study, we determined that culturing iPSC-derived NPCs as three-dimensional (3D) floating neurospheres resulted in increased expression of the neural progenitor cell (NPC) markers,PAX6andNESTIN. Expansion of NPCs in 3D culture methods also resulted in a more homogenous PAX6 expression when compared to 2D culture methods. Furthermore, the 3D propagation method for NPCs resulted in a significant higher expression of the astrocyte markers  GFAPandaquaporin 4(AQP4) in the differentiated cells. Thus, our 3D propagation method could constitute a useful tool to promote NPC homogeneity and also to increase the differentiation potential of iPSC towards astrocytes.

Extracellular vesicles from three dimensional culture of human placental mesenchymal stem cells ameliorated renal ischemia/reperfusion injury

2021 ◽  
pp. 039139882098680
Author(s):  
Xuefeng Zhang ◽  
Nan Wang ◽  
Yuhua Huang ◽  
Yan Li ◽  
Gang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Extracellular Vesicles ◽  
Therapeutic Potential ◽  
Expression Profiles ◽  
Ischemia Reperfusion ◽  
Three Dimensional ◽  
3D Culture ◽  
Placental Mesenchymal Stem Cells ◽  
2D And 3D

Background: Three-dimensional (3D) culture has been reported to increase the therapeutic potential of mesenchymal stem cells (MSCs). The present study assessed the therapeutic efficacy of extracellular vesicles (EVs) from 3D cultures of human placental MSCs (hPMSCs) for acute kidney injury (AKI). Methods: The supernatants from monolayer culture (2D) and 3D culture of hPMSCs were ultra-centrifuged for EVs isolation. C57BL/6 male mice were submitted to 45 min bilateral ischemia of kidney, followed by renal intra-capsular administration of EVs within a 72 h reperfusion period. Histological, immunohistochemical, and ELISA analyses of kidney samples were performed to evaluate cell death and inflammation. Kidney function was evaluated by measuring serum creatinine and urea nitrogen. The miRNA expression profiles of EVs from 2D and 3D culture of hPMSCs were evaluated using miRNA microarray analysis. Results: The 3D culture of hPMSCs formed spheroids with different diameters depending on the cell density seeded. The hPMSCs produced significantly more EVs in 3D culture than in 2D culture. More importantly, injection of EVs from 3D culture of hPMSCs into mouse kidney with ischemia-reperfusion (I/R)-AKI was more beneficial in protecting from progression of I/R than those from 2D culture. The EVs from 3D culture of hPMSCs were more efficient against apoptosis and inflammation than those from 2D culture, which resulted in a reduction in tissue damage and amelioration of renal function. MicroRNA profiling analysis revealed that a set of microRNAs were significantly changed in EVs from 3D culture of hPMSCs, especially miR-93-5p. Conclusion: The EVs from 3D culture of hPMSCs have therapeutic potential for I/R-AKI.

214 Different pluripotency maintenance supplements affect the reprogramming process and pluripotency state of bovine-induced pluripotent stem cells

2020 ◽  
Vol 32 (2) ◽  
pp. 235
Author(s):  
R. Botigelli ◽  
N. Pieri ◽  
B. Bessi ◽  
R. de Castro ◽  
K. Recchia ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Embryoid Body ◽  
Embryoid Bodies ◽  
Cell Reprogramming ◽  
Rt Pcr ◽  
Serum Replacement ◽  
Fetal Fibroblasts ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement ◽  
Pluripotency Maintenance

After the emergence of induced cell reprogramming, achieved through the addition of Yamanaka transcription factors (Oct4, Sox2, Klf4, and cMyc; OSKM) to somatic cells, the number of studies regarding induction and maintenance of pluripotency has increased greatly. The success of bovine iPSCs (biPSCs) was first described by Summer et al. (2011 J. Anim. Sci. 89, 2708-2716; https://doi.org/10.2527/jas.2010-3666); however, investigations on the pluripotent state of biPSCs are still needed because different protocols and characterisation profiles have since been used. The aim of this study was to produce biPSC lines supplemented with different pluripotency maintenance agents to improve self-renewal and pluripotency maintenance. For that, bovine fetal (50 days) fibroblasts (3×104) were transduced with lentivirus harbouring mouse OSKM transcription factors. The cells were further cultured in reprogramming medium (Dulbecco's modified Eagle's medium/F12 KO and 20% KSR (knockout serum replacement)) supplemented with basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), bFGF+2i or LIF+2i (where 2i inhibitors are PD0325901 and CHIR99021). The capacity for cell reprogramming was analysed by colony formation and maintenance after manually and enzymatic passaging and alkaline phosphatase (AP) activity detection; additionally, pluripotency state was assessed by reverse transcription (RT)-PCR (pluripotency biomarkers: OCT4, NANOG, and SOX2; naïve state: STELLA, LIFr, and ESRRb; primed state: OTX2 and FGF5; and mouse (m)OSKM and invitro differentiation assay (embryoid body formation). Statistical analysis was performed using the JMP software (SAS Institute Inc.). All treatments were successful at generating colonies after 28 days of mOSKM transduction, with 32 colonies in bFGF (0.53% efficiency), 21 colonies in bFGF+2i (0.35% efficiency), 5 colonies in LIF (0.08% efficiency), and 3 colonies in LIF+2i (0.05% efficiency) treatments/groups. As an initial pluripotency test, all colonies were positive for AP activity at passage 3. The colonies were cultured for at least 25 passages (±200 days) except for those from the LIF+2i treatment, which were not able to remain viable after 15 passages. Gene expression analysis of the pluripotency (naïve and primed) biomarkers in biPSCs by RT-PCR revealed that colonies from the bFGF treatment were upregulated in NANOG, OCT4, (pluripotency biomarkers), and STELLA (naïve biomarker) (P<0.05) compared with bFGF+2i and LIF groups. There were no differences in expression of SOX2 (pluripotency biomarker gene) and naïve/primed biomarkers (OXT2, LIFr, and ESRRb) (P>0.05). Additionally, the relative abundance of mOSKM was not different between groups (P>0.05). For further pluripotency analysis, biPS colonies were tested for the invitro differentiation assay, and all colonies tested were able to form embryoid bodies. In conclusion, bovine fetal fibroblasts were successfully reprogrammed when using OSKM in all medium tested; however, LIF+2i treatment did not grow beyond 25 passages. Further tests should be performed to determine the pluripotency status of these biPSCs. We acknowledge FAPESP for funding (grant nos. 2012/50533-2, 2015/26816-5, and 2016/16841-2).

scholarly journals Mesenchymal stem cell spheroids: potential cell materials for cell therapy

STEMedicine ◽  
2020 ◽  
Vol 2 (5) ◽  
pp. e67
Author(s):  
Zhongjuan Xu ◽  
Xingzhi Liu ◽  
Yu Wei ◽  
Zhe Zhao ◽  
Junjun Cao ◽  
...  
Keyword(s):  
Regenerative Medicine ◽  
Tissue Injury ◽  
Three Dimensional ◽  
3D Culture ◽  
Research Field ◽  
Three Dimensions ◽  
Spheroid Formation ◽  
Culture Methods ◽  
Differentiation Potential

Mesenchymal stromal/stem cells (MSCs) have been applied in clinical trials with an increasing number in recent years. MSCs showed their great potentials in regenerative medicine for their extensive sources, multilineage differentiation potential, low immunogenicity and self-renewal ability. However, the clinical application of MSCs still confronts many challenges including the requirement of large quantity of cells, low survival ability in vivo and the loss of main original characteristics due to two-dimensional (2D) culture although it is beneficial to cells fast expansion. Three-dimensional (3D) culture artificially creates an environment that permits cells to grow or interact with their surroundings in all three dimensions. Therefore, 3D culture was widely regarded as a more preferable and closer physiological microenvironment for cells growth. Recently, many different 3D spheroid culture methods have been developed to optimize MSCs biological characteristics to meet the demand of regenerative medicine. In this review, we comprehensively discussed the merits and demerits of different spheroid formation methods, expounded the mechanisms of spheroid formation and its microenvironment, and illustrated their optimized biological functions and the pre-clinical applications in various tissue injury and regeneration. In the end, we prospected the trends of this research field and proposed the key problems needed to be solved in the future.

scholarly journals Establishment of Bovine-Induced Pluripotent Stem Cells

2021 ◽  
Vol 22 (19) ◽  
pp. 10489
Author(s):  
Yue Su ◽  
Ling Wang ◽  
Zhiqiang Fan ◽  
Ying Liu ◽  
Jiaqi Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Colony Morphology ◽  
Significant Advance ◽  
Serum Replacement ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement

Pluripotent stem cells (PSCs) have been successfully developed in many species. However, the establishment of bovine-induced pluripotent stem cells (biPSCs) has been challenging. Here we report the generation of biPSCs from bovine mesenchymal stem cells (bMSCs) by overexpression of lysine-specific demethylase 4A (KDM4A) and the other reprogramming factors OCT4, SOX2, KLF4, cMYC, LIN28, and NANOG (KdOSKMLN). These biPSCs exhibited silenced transgene expression at passage 10, and had prolonged self-renewal capacity for over 70 passages. The biPSCs have flat, primed-like PSC colony morphology in combined media of knockout serum replacement (KSR) and mTeSR, but switched to dome-shaped, naïve-like PSC colony morphology in mTeSR medium and 2i/LIF with single cell colonization capacity. These cells have comparable proliferation rate to the reported primed- or naïve-state human PSCs, with three-germ layer differentiation capacity and normal karyotype. Transcriptome analysis revealed a high similarity of biPSCs to reported bovine embryonic stem cells (ESCs) and embryos. The naïve-like biPSCs can be incorporated into mouse embryos, with the extended capacity of integration into extra-embryonic tissues. Finally, at least 24.5% cloning efficiency could be obtained in nuclear transfer (NT) experiment using late passage biPSCs as nuclear donors. Our report represents a significant advance in the establishment of bovine PSCs.

scholarly journals Mbd3/NuRD is a Key Inhibitory Module During the Induction and Maintenance of Naïve Pluripotency

2015 ◽  
Author(s):  
Asaf Zviran ◽  
Yoach Rais ◽  
Nofar Mor ◽  
Noa Novershtern ◽  
Jacob H Hanna
Keyword(s):  
Pluripotent Stem Cell ◽  
Primordial Germ Cells ◽  
Induced Pluripotent Stem Cell ◽  
Major Pathway ◽  
Serum Replacement ◽  
Epiblast Stem Cells ◽  
Induced Pluripotent ◽  
Knockout Serum Replacement ◽  
Formation Efficiency

Our group has published a study on induced pluripotent stem cell (iPSC) reprogramming (Rais et al. Nature 20131) that reached the following conclusions: a) Mbd3/NuRD is a repressor of inducing naïve pluripotency from mouse Epiblast stem cells (EpiSCs), primordial germ cells (PGCs), murine somatic cells and human secondary fibroblasts; b) Up to 100% iPSC formation efficiency can be achieved via optimized Mbd3/NuRD depletion, in concert with optimized OKSM delivery and naïve pluripotency conditions (2i supplement applied only after 48 hours, human LIF, hypoxia and Vitamin C containing Knockout serum replacement)1. This represented the first proof for deterministic/near-deterministic iPSC reprogramming, and highlighted a previously unappreciated role for Mbd3/NuRD in hampering the re-establishment of pluripotency. Recent reports have seemingly provided contradictory results and attempted to dispute our iPSC efficiency quantifications and/or the role of Mbd3/NuRD in blocking reprogramming2,3. Here we provide a detailed response to these reports based on extended discussions and providing new data. The synthesis presented herein disagrees with claims made by Silva, Hendrich, Bertone and colleagues2,3, and reconfirms that Mbd3/NuRD is a major pathway that inhibits the maintenance and induction of pluripotency1. Further, we foresee that its controlled manipulation is likely to become an integral pathway for inducing and maintaining naïve pluripotency in a variety of species.

scholarly journals Microfluidic-Based Droplets for Advanced Regenerative Medicine: Current Challenges and Future Trends

Biosensors ◽  
2021 ◽  
Vol 12 (1) ◽  
pp. 20
Author(s):  
Hojjatollah Nazari ◽  
Asieh Heirani-Tabasi ◽  
Sadegh Ghorbani ◽  
Hossein Eyni ◽  
Sajad Razavi Bazaz ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Regenerative Medicine ◽  
Cell Therapy ◽  
Large Scale ◽  
Three Dimensional ◽  
3D Culture ◽  
Cell Encapsulation ◽  
Cost Effective ◽  
Culture Methods

Microfluidics is a promising approach for the facile and large-scale fabrication of monodispersed droplets for various applications in biomedicine. This technology has demonstrated great potential to address the limitations of regenerative medicine. Microfluidics provides safe, accurate, reliable, and cost-effective methods for encapsulating different stem cells, gametes, biomaterials, biomolecules, reagents, genes, and nanoparticles inside picoliter-sized droplets or droplet-derived microgels for different applications. Moreover, microenvironments made using such droplets can mimic niches of stem cells for cell therapy purposes, simulate native extracellular matrix (ECM) for tissue engineering applications, and remove challenges in cell encapsulation and three-dimensional (3D) culture methods. The fabrication of droplets using microfluidics also provides controllable microenvironments for manipulating gametes, fertilization, and embryo cultures for reproductive medicine. This review focuses on the relevant studies, and the latest progress in applying droplets in stem cell therapy, tissue engineering, reproductive biology, and gene therapy are separately evaluated. In the end, we discuss the challenges ahead in the field of microfluidics-based droplets for advanced regenerative medicine.

scholarly journals Development of a 3D human osteoblast cell culture model for studying mechanobiology in orthodontics

2020 ◽  
Vol 42 (4) ◽  
pp. 387-395
Author(s):  
Damien Brezulier ◽  
Pascal Pellen-Mussi ◽  
Sylvie Tricot-Doleux ◽  
Agnès Novella ◽  
Olivier Sorel ◽  
...  
Keyword(s):  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Gene Expression Profiles ◽  
Tissue Response ◽  
Cell Culture Model ◽  
Glucose Levels ◽  
Culture Model ◽  
Polymerase Chain ◽  
Adjustable Weight

Summary Objectives Mechanobiology phenomena constitute a major element of the cellular and tissue response during orthodontic treatment and the implantation of a biomaterial. Better understanding these phenomena will improve the effectiveness of our treatments. The objective of this work is to validate a model of three-dimensional (3D) culture of osteoblasts to study mechanobiology. Materials and methods The hFOB 1.19 cell line was cultured either traditionally on a flat surface or in aggregates called spheroids. They were embedded in 0.8% low-melting agarose type VII and placed in a polyethylene terephthalate transwell insert. Compressive forces of 1 and 4 g/cm2 were applied with an adjustable weight. Proliferation was evaluated by measuring diameters, monitoring glucose levels, and conducting Hoechst/propidium iodide staining. Enzyme-linked immunosorbent assays focusing on the pro-inflammatory mediators interleukin (IL)-6 and IL-8 and bone remodelling factor osteoprotegerin were performed to evaluate soluble factor synthesis. quantitative reverse transcription-polymerase chain reaction was performed to evaluate bone marker transcription. Results The 3D model shows good cell viability and permits IL dosing. Additionally, three gene expression profiles are analysable. Limitations The model allows analysis of conventional markers; larger exploration is needed for better understanding osteoblast mechanobiology. However, it only allows an analysis over 3 days. Conclusion The results obtained by applying constant compressive forces to 3D osteoblastic cultures validate this model system for exploring biomolecule release and analysing gene transcription. In particular, it highlights a disturbance in the expression of markers of osteogenesis.

scholarly journals Zebrafish skeletal muscle cell cultures: Monolayer to three-dimensional tissue engineered collagen constructs

2020 ◽  
Author(s):  
K.K Vishnolia ◽  
N.R.W Martin ◽  
D.J Player ◽  
E Spikings ◽  
M.P Lewis
Keyword(s):  
Skeletal Muscle ◽  
Mrna Expression ◽  
Expression Profiles ◽  
Three Dimensional ◽  
3D Culture ◽  
Morphological Characteristics ◽  
Hypertrophic Growth ◽  
3D Cultures

AbstractZebrafish (Danio rerio) are a commonly used model organism to study human muscular myopathies and dystrophies. To date, much of the work has been conducted in vivo due to limitations surrounding the consistent isolation and culture of zebrafish muscle progenitor cells (MPCs) in vitro and the lack of physiologically relevant models.Here we report a robust, repeatable, and cost-effective protocol for the isolation and culture of zebrafish MPCs in conventional monolayer (2D) and have successfully transferred these cells to 3D culture in collagen based three-dimensional (3D) tissue-engineered constructs. Zebrafish MPC’s cultured in 2D were consistently reported to be Desmin positive reflecting their muscle specificity, with those demonstrating Desmin positivity in the 3D cultures. In addition, mRNA expression of muscle markers specific for proliferation, differentiation and maturation measured from both monolayer and 3D cultures at appropriate developmental stages were found consistent with previously published from other species in vitro and in vivo muscle data.Collagen constructs seeded with zebrafish MPC’s were initially characterised for optimal seeding density, followed by macroscopic characterisation (three-fold contraction) of the matrix. Direct comparison between the morphological characteristics (proportion of cells) and gene expression profiles of cells cultured in collagen constructs revealed higher maturation and differentiation compared to monolayer cultures. In this regard, cells embedded in 3D collagen constructs revealed higher fusion index, Desmin positivity, hypertrophic growth, myotube maturity and myogenic mRNA expression when compared to in monolayer.In conclusion, these methods and models developed herein will facilitate in vitro experiments, which would complement in vivo zebrafish studies used to investigate the basic developmental, myopathies and dystrophies in skeletal muscle cells.

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