bFGF+ MSC bFGF- MSC

Activation of endogenous regenerative mechanisms leading to bone formation

by bFGF-selected MSC seeded on scaffold and ectopically implanted in vivo.

Gaetani M (1, 2, 3), Tasso R (3), Molino E (3), Cattaneo A (4), Monticone M (3), Bachi A (4), Conaldi PG (1, 2), Cancedda R (3)

(1) Ri.MED Foundation, Palermo (PA), Italy(2) Regenerative Medicine and Biomedical Technologies Unit, University of Pittsburgh Medical Center, ISMETT, Palermo (PA), Italy(3) Department of Oncology, Biology and Genetics, University of Genoa and National Cancer Research Institute, Genova (GE), Italy(4) Biomolecular Mass Spectrometry Unit, Division of Genetics & Cell Biology, San Raffaele Scientific Institute, Milano (MI), Italy

Bibliography

Tasso R*, Gaetani M* et al. “Implanted Mesenchymal Stem Cells Selected by Basic Fibroblast Growth Factor Induce a Host Response Leading to Bone Regeneration.” Biomaterials 2012;33:2086-2096 [ * equal contributors ]

Prockop DJ. “Repair of tissues by adult stem/progenitor cells (MSCs): controversies, myths, and changing paradigms.” Mol Ther 2009;17:939-946

Caplan AI, Correa D. “The MSC: An Injury Drugstore.” Cell Stem Cell 2011;9:11-15

Abstract

Summary of Major Results

The view depicting bone marrow (BM)-derived mesenchymal stem cells (MSC) as a uniform

population differentiating into new-tissue builder cells is evolving toward the concept of a

heterogeneous population of stem/progenitor cells secreting bioactive molecules and trophicfactors, and contributing to establish an on-site regenerative microenvironment. We report that

in an ectopic bone formation model the intrinsic MSC capability to activate endogenous

regenerative mechanisms is critically dependent on the commitment degree of the implanted

MSC. We demonstrate that the presence of bFGF in the culture medium during mouse MSC

expansion in vitro is the key factor for the selection of subpopulations at an early progenitor level and that induce host regenerative responses. A novel strategy, developed by combining

quantitative SILAC LC-MS/MS -based proteomics of cultured cells and conditioned media

(CM) together with gene expression profiling, disentangled the major role of MSC in

modulating the microenvironment toward the damage resolution. Instead of proteomics of cell

lysates, proteomics of CM interestingly provided corresponding results with transcriptomics, so proving that the most significant biological processes favored by the bFGF treatment were

carried out by secreted factors. In particular, “immune response", “inflammatory response”,

“response to wounding” and “chemotaxis” were upregulated by bFGF+ MSC. Therefore, these

processes appear to be major actors within the host regenerative responses, that lead to bone

neo-formation, generated by ectopic implants of ceramic scaffolds seeded with bFGF+ MSC.

(~2.5 106 cells)Fig.1

Fig.2

The presence of bFGF in the culture medium during cell expansion in vitro influences the ability of MSC seeded onto ceramic scaffolds and ectopically implanted in vivo to induce endogenous regenerative processes, so leading to the formation of new bone tissue.

(A) A representative cartoon of the experimental setting using the ectopic bone formation mouse model and mouse GFP+ BM-derived MSC seeded onto ceramic scaffolds. (B) Hematoxylin/Eosin (H/E) and Toluidine Blue (TB) stainings showed cartilage (arrow) and undifferentiated fibrous tissue (asterisk) within the pores of the scaffolds seeded with bFGF+ and bFGF- MSC, at day 30 (left panel) an 60 (right panel) after implantation. Anti-GFP immunostaining at day 60 showed endochondral ossification by recruited host cells in the case of bFGF+ MSC, while direct ossification occurred with untreated MSC.

Aknowledgements

M.G. was supported by a fellowship in regenerative medicine / tissue engineering from the McGowan Institute for Regenerative Medicine of the University of Pittsburgh, within a research program directed by Prof. R. Cancedda and established at ISMETT, in collaboration with the Regenerative Medicine Unit that he heads at the University of Genoa and at the National Cancer Research Institute.

A

B

Table 1

Comparative characterization of MSC cultured both with and without bFGF established a relationship between donor cell commitment and regenerative responses to cell implantation.

(A) Normalized percentage of CFU-f of 15x106 BM-nucleated cells cultured for 2 weeks, with or without bFGF. Results are averaged from 6 independent experiments. ALP -positive and -negative colonies are represented respectively by black and grey columns. (B) Comparative flowcytometric analysis of bFGFtreated and untreated MSC. Areas under black lines identify cells reacting with specific antigens. Areas under grey lines indicate interactions of cells with non-reactive immunoglobulin of the same isotype.

A B

bFGF selects MSC subpopulations with:

� increased clonogenic potential

� early progenitor phenotype

Fig.3“LIGHT” (L)

Arg + Lys

“HEAVY”(H)

Arg + Lys

SILAC (Stable Isotope Labeling by Aminoacid in Cell Culture)-

-based PROTEOMICS of conditioned media and cell lysate

A new experimental strategy developed by combining quantitative comparative –OMICS sciences

Bioinformatics

and

Functional Data

Analyses

TRANSCRIPTOMICS:

GeneChip

microarray analysis

(Affimetrix-based)

YES1.7E-114.121Chemotaxis

YES8.2E-127.237Response to Wounding

YES8.4E-136.031Inflammatory Response

NO6.6E-146.232Leukocyte Activation

YES4.1E-3113.670Immune Responses

CorrespondenceP-value%CountGO biological process

Proteomic AnalysisGene Expression AnalysisSystem Gene Category

The most statistically significant biological processes upregulated by MSC upon bFGF treatment were carried out majorly through secreted activities modulating the cell microenvironment.

GO cluster analysis of upregulated (525 total) but not downregulated (109) transcripts showed correspondence with biological processes revealed by proteomics of secreted factors increased by bFGFtreatment (shown in Fig.4). Arrows indicate matching categories.

bFGF+ MSC bFGF- MSC

+ bFGFor

no bFGF

Porous ceramic scaffold

GFP+ MSC Subcutaneous implant

bFGF+ MSC bFGF- MSC bFGF+ MSC bFGF- MSC

Quantitative proteomics revealed factors differentially secreted by FGF+ MSC versus those secreted by FGF- MSC that are able to modulate the cell microenvironment toward the activation of specific biological processes simultaneously involved in tissue regeneration.

Diagram depiction (Cytoscape 2.8 software) of major differentially secreted proteins, shown as small circles color-coded by their FGF+ MSC versus FGF- MSC final average ratio, as indicated by the color legend (right); positive and negative bFGF regulation are shown in panels A and B, respectively. The gene ontology categories clustered using bioinformatics functional data analysis are represented by yellow hexagons. The arrows indicate the correspondence between the major biological functions of the identified secreted factors and those clustered by genome wide expression analysis (shown in Table 1). The light blue text box (up left) describes major hints of the newly developed proteomics strategy.

� The endochondral new bone formation obtained by implanting scaffolds seeded with bFGF+ MSC was produced by host cells and by the activation endogenous regenerative processes.

� bFGF treated MSC showed higher clonogenic potential and were enriched of early progenitor cells.

� The endogenous processes putatively activated as simultaneous “switches” to turn on the new tissue formation into the implanted ceramic scaffolds seeded with MSC were: response of innate immunity, inflammatory response, response to wounding and chemotaxis.

� These host biological processes are simultaneously triggered by factors secreted by bFGF+ MSC modulating the microenvironment so as to turn on the regenerative “switches”.

� Major soluble markers differentially produced by MSC upon bFGF treatment were identified.

Final Remarks and Extended Impacts� We experimentally demonstrated: (1) the new paradigm about implanted MSC and their induction of

regenerative endogenous responses by secreting bioactive molecules and establishing a regenerative microenvironment, rather than to directly differentiate into the injured tissue; (2) that this ability is linked to MSC commitment/stemness degree; (3) that specific culture conditions can drive cell population heterogeneity toward the enrichment of appropriately committed cells in vitro.

� We developed a new comprehensive strategy using advanced –OMICS sciences that is able to identify molecular and functional biomarker activities of MSC cultures with regenerative properties.

Fig.4• Two inverse SILAC labelings (D and R,

i.e. bFGF- and bFGF+ MSC respectively

in L and H cultures, and viceversa)

were adopted to reach confident mass

spectrometric protein identification;

• A total of 804 and 818 proteins of CM

were identified in D and R, respectively;

• Relative differences in proteins

released by bFGF+ MSC versus bFGF-

MSC were quantified using H/L ratios;

• Final quantification of identified

proteins in the conditioned media

showed significant changes: 35 and 32

factors respectively increased and

decreased upon bFGF treatment;

• Appreciable levels of experimental

reproducibility, both technical (R2>0.98)

and biological (R2>0.85), was reached;

• Bioinformatics and functional data

analyses defined 4 major endogenous

biological processes supported by

secretive activity of bFGF+ MSC rather

than bFGF- MSC.

A

B