Extracellular matrix microarrays to study inductive signaling for endoderm specification

Malta, David F. Braga; Reticker-Flynn, Nathan E.; Silva, C.L. da; Fleming, Heather E.; Zaret, Kenneth S.; Underhill, Gregory H.

doi:10.1016/j.actbio.2016.02.014

Cited by 23 publications

(22 citation statements)

References 42 publications

(47 reference statements)

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“…In this reductionist approach, the synergistic impact of growth factors and ECM proteins and their coordinated signalling pathways in the keratinocytes is overlooked. Others have shown that the combination of three matrix proteins can have a synergistic effect 63–65 . Flaim et al .…”

Section: Discussionmentioning

confidence: 99%

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

Wong

LeGrand

Kinnear

et al. 2019

Sci Rep

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The long-term expansion of keratinocytes under conditions that avoid xenogeneic components (i.e. animal serum- and feeder cell-free) generally causes diminished proliferation and increased terminal differentiation. Here we present a culture system free of xenogeneic components that retains the self-renewal capacity of primary human keratinocytes. In vivo the extracellular matrix (ECM) of the tissue microenvironment has a major influence on a cell’s fate. We used ECM from human dermal fibroblasts, cultured under macromolecular crowding conditions to facilitate matrix deposition and organisation, in a xenogeneic-free keratinocyte expansion protocol. Phospholipase A2 decellularisation produced ECM whose components resembled the core matrix composition of natural dermis by proteome analyses. Keratinocytes proliferated rapidly on these matrices, retained their small size, expressed p63, lacked keratin 10 and rarely expressed keratin 16. The colony forming efficiency of these keratinocytes was enhanced over that of keratinocytes grown on collagen I, indicating that dermal fibroblast-derived matrices maintain the in vitro expansion of keratinocytes in a stem-like state. Keratinocyte sheets formed on such matrices were multi-layered with superior strength and stability compared to the single-layered sheets formed on collagen I. Thus, keratinocytes expanded using our xenogeneic-free protocol retained a stem-like state, but when triggered by confluence and calcium concentration, they stratified to produce epidermal sheets with a potential clinical use.

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Section: Discussionmentioning

confidence: 99%

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

Wong

LeGrand

Kinnear

et al. 2019

Sci Rep

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“…The ability to systematically explore combinations of microenvironmental stimuli that includes CCN 1-6 proteins on tumor cell behavior could be considered with an approach reported by Malta et al (2016). They constructed an array with 741 distinct combinations of 38 different ECM components to study embryonic stem cells and the derived endoderm progenitors.…”

Section: Summary and Perspectivementioning

confidence: 99%

CCN family of proteins: critical modulators of the tumor cell microenvironment

Yeger

Perbal

2016

J. Cell Commun. Signal.

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The CCN family of proteins consisting of CCN1 (Cyr61), CCN2 (CTGF), CCN3 (NOV), CCN4 (WISP-1), CCN5 (WISP-2) and CCN6 (WISP-3) are considered matricellular proteins operating essentially in the extracellular microenvironment between cells. Evidence has also been gradually building since their first discovery of additional intracellular roles although the major activity is triggered at the cell membrane. The proteins consist of 4 motifs, a signal peptide (for secretion} followed consecutively by the IGFBP, VWC, TSP1 and CT (C-terminal cysteine knot domain) motifs, which signify their potential binding partners and functional connections to a variety of key regulators of physiological processes. With respect to cancer it is now clear that, whereas certain members can facilitate tumor behavior and progression, others can competitively counter the process. It is therefore clear that the net outcome of biological interactions in the matrix and what gets signaled or inhibited can be a function of the interplay of these CCN 1-6 proteins. Because the CCN proteins further interact with other key proteins, like growth factors in the matrix, the balance is not only important but can vary dynamically with the physiological states of tumor cells and the surrounding normal cells. The tumor niche with its many cell players has surfaced as a critical determinant of tumor behavior, invasiveness, and metastasis. It is in this context that CCN proteins should be investigated with the potential of being recognized and validated for future therapeutic approaches.

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“…Indeed, we recently found that ligands were a major driver of resistance to HER2-targeted inhibitors, but that these effects could be modulated by the ECM 5 . A variety of cells, including primary cells and cell lines derived from different cell types including lung, bladder, prostate, breast, and pancreas, and iPS stem cells, have been successfully cultured on the MEMA platform (see examples in 5,7,14 ). The use of different stains allows for the readout of multiple cellular endpoints, including cell growth, differentiation, and metabolism.…”

Section: Discussionmentioning

confidence: 99%

“…Since the microenvironments are simple combinations, it is straightforward to identify proteins that drive major phenotypic changes in cells. MEMAs have been used successfully to identify factors that influence multiple cellular phenotypes, including those that drive cell fate decisions and response to therapy 4–7 . These responses can be validated in simple 2-D experiments and can then be assessed under conditions that more fully recapitulate the complexity of the tumor microenvironment.…”

Section: Introductionmentioning

confidence: 99%

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

Smith

Devlin

Kilburn

et al. 2019

JoVE

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Understanding the impact of the microenvironment on the phenotype of cells is a difficult problem due to the complex mixture of both soluble growth factors and matrix-associated proteins in the microenvironment in vivo. Furthermore, readily available reagents for the modeling of microenvironments in vitro typically utilize complex mixtures of proteins that are incompletely defined and suffer from batch to batch variability. The microenvironment microarray (MEMA) platform allows for the assessment of thousands of simple combinations of microenvironment proteins for their impact on cellular phenotypes in a single assay. The MEMAs are prepared in well plates, which allows the addition of individual ligands to separate wells containing arrayed extracellular matrix (ECM) proteins. The combination of the soluble ligand with each printed ECM forms a unique combination. A typical MEMA assay contains greater than 2,500 unique combinatorial microenvironments that cells are exposed to in a single assay. As a test case, the breast cancer cell line MCF7 was plated on the MEMA platform. Analysis of this assay identified factors that both enhance and inhibit the growth and proliferation of these cells. The MEMA platform is highly flexible and can be extended for use with other biological questions beyond cancer research.

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Extracellular matrix microarrays to study inductive signaling for endoderm specification

Cited by 23 publications

References 42 publications

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

In Vitro Expansion of Keratinocytes on Human Dermal Fibroblast-Derived Matrix Retains Their Stem-Like Characteristics

CCN family of proteins: critical modulators of the tumor cell microenvironment

Using Microarrays to Interrogate Microenvironmental Impact on Cellular Phenotypes in Cancer

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