3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Kabiri, Mahboubeh; Kul, Betul; Lott, William B.; Futrega, Kathryn; Ghanavi, Parisa; Upton, Zee; Doran, Michael

doi:10.1016/j.bbrc.2012.01.017

Cited by 64 publications

(60 citation statements)

References 29 publications

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“…61 The results are in line with a recent study of Kabiri et al, who observed upregulated BMP-2 expression levels in BM-MSCs cultured as 3D-microaggregates when compared with 2D-monolayer enhancing osteogenic differentiation. 62 Neither the 3D environment influenced the gene expression level of the osteogenic target gene ALPL, nor did we detect an increase of the corresponding protein activity. ALP activity levels were only significantly enhanced by the use of osteogenic supplements under 2D conditions on day 21 but already elevated after 7 days in combination with 3D culture increasing over the course of time (14, 21 days).…”

Section: Discussionmentioning

confidence: 57%

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Rauh

Jacobi

Stiehler

2015

Tissue Engineering Part C: Methods

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The principles of tissue engineering (TE) are widely used for bone regeneration concepts. Three-dimensional (3D) cultivation of autologous human mesenchymal stromal cells (MSCs) on porous scaffolds is the basic prerequisite to generate newly formed bone tissue. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is a specific and sensitive analytical tool for the measurement of mRNA-levels in cells or tissues.For an accurate quantification of gene expression levels, stably expressed reference genes (RGs) are essential to obtain reliable results. Since the 3D environment can affect a cell's morphology, proliferation, and gene expression profile compared with two-dimensional (2D) cultivation, there is a need to identify robust RGs for the quantification of gene expression. So far, this issue has not been adequately investigated. The aim of this study was to identify the most stably expressed RGs for gene expression analysis of 3D-cultivated human bone marrow-derived MSCs (BM-MSCs). For this, we analyzed the gene expression levels of n = 31 RGs in 3D-cultivated human BM-MSCs from six different donors compared with conventional 2D cultivation using qRT-PCR. MSCs isolated from bone marrow aspirates were cultivated on human cancellous bone cube scaffolds for 14 days. Osteogenic differentiation was assessed by cell-specific alkaline phosphatase (ALP) activity and expression of osteogenic marker genes. Expression levels of potential reference and target genes were quantified using commercially available TaqMan Ò assays. mRNA expression stability of RGs was determined by calculating the coefficient of variation (CV) and using the algorithms of geNorm and NormFinder. Using both algorithms, we identified TATA box binding protein (TBP), transferrin receptor (p90, CD71) (TFRC), and hypoxanthine phosphoribosyltransferase 1 (HPRT1) as the most stably expressed RGs in 3D-cultivated BMMSCs. Notably, genes that are routinely used as RGs, for example, beta actin (ACTB) and ribosomal protein L37a (RPL37A), were among the least stable genes. We recommend the combined use of TBP, TFRC, and HPRT1 for the accurate and robust normalization of qRT-PCR data of 3D-cultivated human BM-MSCs.

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Section: Discussionmentioning

confidence: 57%

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Rauh

Jacobi

Stiehler

2015

Tissue Engineering Part C: Methods

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“…Similar to previous studies [337,342,351,353], MSC tri--lineage was inducible in 3D spheroids using the microwell platform. …”

Section: Manufacture Of 3d Msc Spheroid Cultures and Characterisationsupporting

confidence: 62%

“…Microtissues are appealing building blocks in tissue engineering applications [334,341,351,395,409], including the manufacture of cartilage microtissues from either mesenchymal stem/stromal cells (MSC) [337] or articular chondrocytes [395]. The fundamental motivation for using a microwell platform to manufacture cartilage microtissues is its capacity to reproducibly generate hundreds of uniform tissue building blocks.…”

Section: Introductionmentioning

confidence: 99%

Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

Futrega¹

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KeywordsAIM 2: Utilization of the high throughput microwell platform to manufacture HSPC/MSC spheroids to improve the efficacy of direct BM transplantation.AIM 3: Development of an improved microwell platform that retains spheroids within discrete microwells throughout culture. AIM 4:Characterization of HSPC surface marker change in response to microwell material. DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHEROID CULTURES iiiChapter 2 describes the development of a high throughput HSPC/MSC 3D spheroid co--culture embodied in AIM 1. I developed a high throughput polydimethylsiloxane (PDMS) microwell platform that enabled the manufacture of thousands of uniform 3D multicellular co--culture spheroids. Spheroids were assembled such that each contained 25--to--400 BM--derived MSC and 10 CB--derived CD34 + cells (a cell population enriched for HSPC). The outcomes from this Chapter guided the design of the subsequent three chapters. The specific outcomes and subsequent study results were as follows:Chapter 3: As described in Chapter 2, only modest improvements in expansion could be achieved by co--culturing HSPC in 3D MSC spheroids using a microwell platform. We reasoned that the 3D spheroids could, however, function as a biologically active anchor to improve direct BM transplantation outcomes. In this Chapter, we contrasted transplantation methods, including the use of MSC suspensions and 3D multicellular spheroids to modulate human CB--derived CD34 + cell retention within the BM cavity of NOD/SCID gamma (NSG) mice. Analysis revealed that the BM of injected femurs in some animals was saturated with human cells, while distal haematopoietic tissue engraftment was poor. These data show, for the first time, that retention of human haematopoietic cells within the BM enhances local engraftment at the expense of systemic engraftment.Chapter 4: As identified in Chapter 2, improvements in 3D spheroid co--cultures will require that the microwell platform is compatible with repeated medium exchange or dilution feeding. Multiple partial or complete medium exchanges can displace spheroids from discrete microwells, and this can result in either the loss of spheroids from culture, or spheroid amalgamation when displaced microtissues fall into common microwells. In this Chapter, I describe the first microwell platform that incorporates a mesh to retain microtissues within discrete microwells. We called this platform the microwell--mesh. We show that bonding a nylon mesh with an appropriate pore size over the microwell openings allows single cells to pass through the mesh into the microwells during the seeding process, but subsequently retains assembled microtissues within discrete microwells.Chapter 5: As identified in Chapter 2, the presence of PDMS, the material used to fabricate the microwell platform, altered CD38 surface expression on CB--derived CD34 + cells. Previous reports have shown that inhibition of retinoic acid signaling down--regulated CD38 iv DEVICE AND APPLICATION DEVELOPMENT FOR HSPC AND MSC 3D SPHE...

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“…Ethical approval for this study was granted through the Mater Health Services Human Research Ethics Committee and the Queensland University of Technology in accordance with the Australian National Health and Medical Research Council's Statement on Ethical Conduct in Research Involving Humans. Samples were diluted with phosphate-buffered saline (PBS) and mononuclear cells were enriched via density gradient centrifugation as described previously [23,24]. MSC were expanded in low-glucose DMEM (Gibco) containing 10% FBS and 1% P/S, and then sub-cultured in DMEM/F12 supplemented with 5% FBS and 1% P/S for one passage prior to use in migration assays to enable cell adaption to the base medium used in the MCF-7 monocultures and planned migration assays.…”

Section: Cell Isolation and Culturementioning

confidence: 99%

In Vitro Assessment of Migratory Behavior of Two Cell Populations in a Simple Multichannel Microdevice

et al. 2013

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Recent literature suggests that mesenchymal stem/stromal cells (MSC) could be used as Trojan Horses to deliver "death-signals" to cancer cells. Herein, we describe the development of a novel multichannel cell migration device, and use it to investigate the relative migration rates of bone marrow-derived MSC and breast cancer cells (MCF-7) towards each other. Confluent monolayers of MSC and MCF-7 were established in adjacent chambers separated by an array of 14 microchannels. Initially, culture chambers were isolated by air bubbles (air-valves) contained within each microchannel, and then bubbles were displaced to initiate the assay. The MCF-7 cells migrated preferentially towards MSC, whilst the MSC did not migrate preferentially towards the MCF-7 cells. Our results corroborate previous literature that suggests MSC migration towards cancer cells in vivo is in response to the associated inflammation rather than directly to signals secreted by the cancer cells themselves. OPEN ACCESSProcesses 2013, 1 350

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3D mesenchymal stem/stromal cell osteogenesis and autocrine signalling

Cited by 64 publications

References 29 publications

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Identification of Stable Reference Genes for Gene Expression Analysis of Three-Dimensional Cultivated Human Bone Marrow-Derived Mesenchymal Stromal Cells for Bone Tissue Engineering

Device and application development for haematopoietic stem and progenitor cell (HSPC) and mesenchymal stromal cell (MSC) 3D spheroid cultures

In Vitro Assessment of Migratory Behavior of Two Cell Populations in a Simple Multichannel Microdevice

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