Depicting the Bone Marrow Microenvironment Towards the Ex-vivo Expansion of Human Stem Cells David Braga Malta SCBL-IST LMRT-MIT S TEM C ELL B IOENGINEERING L ABORATORY Cláudia Lobato da Silva Joaquim Sampaio Cabral Sangeeta N Bhatia Gregory Underhill Bioengineering Systems

 Combinatorial ex vivo microenvironment to expand adult stem cells Develop: Study:  Bone marrow niche  Cell-cell interactions  Cell-chemical cues interactions  Cell-ECM interactions NIH Stem Cell Report 2001 Stem Cell Expansion  Adult Stem Cells have been used in the clinic for 50 years - BMT  Lack of compatible donors or limited cell numbers motive the need for expansion of these cells  Bone Marrow Niche  Hematopoietic Stem Cells  Mesenchymal Stem Cells

Bone Marrow Niche NIH Stem Cell Report 2001 Cellular Constituents - Hematopoietic Stem Cells (HSC) - Mesenchymal Stem Cells (MSC) Extracellular Matrix (ECM) Components -Osteopontin -Collagens Chemical Cues – Growth Factors - SCF - LIF - …

Deconstructing the Bone Marrow Niche Step I -Study MSC expansion/biology and ECM signaling -Optimization and characterization of the stromal layer (MSC) for HSC co-culture Step II - ECM-MSC soluble factor screen Step III - ECM-MSC-HSC interactions and the niche characterization Step IV - Ex-vivo expansion of MSC and HSC Wilson & Trumpp Nat Rev Immunology 2006

ECM Array Platforms Mei, Y. et al, small, 2008 “Microscale Direct Writing (MDW)” technology for generating two-component single-cell microarrays LaBarge, M.A., Integr. Biol., 2009 Combinatorial Microenvironments for understanding mammary progenitor cell fate decisions Flaim et al. Nature Methods, 2005 Large-scale combinatorial extracellular matrix screening arrays

Existing Array Technology Flaim et al. Nature Methods, 2005; Flaim et al. Stem Cells and Development, 2007

MSC-ECM Pilot Studies Preliminary data Protocol Optimization – Seeding Densities – Readouts Development of a Pipeline for the 2 nd generation MSC spots stained for proliferation Cells stained with Hoechst (blue) and BrdU (green)

ECM-MSC Adhesion

2 nd generation ECM Arrays Design Criteria – Blind screen – Large screen – All commercially available ECM molecules – 38 molecules – Two controls – Biological and Seeding ~800 ECM combination – All parwise combinations of the 38 molecules

Expanded ECM Arrays Collagen I Collagen II Collagen III Collagen IV Collagen V Collagen VI Fibronectin Laminin Merosin (laminin alpha2) Vitronectin Tenascin-C Tenascin-R Chondroitin Sulfate Aggrecan Decorin Elastin Keratin Mucin Superfibronectin Agrin F-Spondin Nidogen-1 Nidogen-2 Heparan Sulfate Hyaluronic Acid Biglycan Decorin Galectin 1 Galectin 3 Galectin 3c Galectin 4 Galectin 8 Testican 1/SPOCK1 Testican 2/SPOCK2 Osteopontin SPARC/Osteonectin Thrombospondin-4 Brevican ECM Molecules Incorporated Brevican/Chondroitin Sulfate

Array Fabrication and Analysis PA Gel Fab Matrix Spotting Cell Seeding Imaging Analysis

Data Analysis

MSC Adhesome Cell Numbers

Statistical Tests Cell Numbers Average per combination Outliers removed Each combination is presented 5 times Outliers – 1 std dev apart from combination mean 3% of outliers (22 combinations out of 800) Robustness of the internal replicates

Replicate Strength Day 1 MSC Adhesome Rep 1 Day 1 MSC Adhesome Rep 2 All SpotsAverage Independent experiments align in the x=y axis Top Adhesion Combinations: Galectin-1 Fibronectin Fibronectin Collagen III Galectin-1 Vitronectin Fibronectin Collagen II Aggrecan Collagen VI Galectin-4 Brevican Fibronectin

MSC Experiments in Data Analysis Seeding Conditions: – Influence of serum concentration – Initial cell density Proliferation vs Differentiation Tracking the in vitro differentiation: – Bone – Cartilage – Adipose Tissue

Next Steps ECM-MSC-Soluble Factor Study – Effect of the main effectors of the main cellular pathways on cell fate in a cross talk with ECM environment Study the mechanism behind the ECM signaling on cell fate – Knockout studies

3 rd Generation ECM array 96 well format Enable integration with HTS platforms Drug/molecule screening Study rare cell types

1 96 well plate well fits a 6 by 6 grid 1 96 well plate accommodates 3456 spots

ECM array as a co-culture system ECM-MSC-HSC studies – Co-culture in the array set up – Study the mechanism of HSC induced expansion by ECM-MSC Study ECM induced phenotypes in rare cell types – Primary cardiac progenitor cells – ES derived endoderm progenitor cells Continue to develop the technological platform

LMRT – Sangeeta N. Bhatia – Gregory Underhill – Nathan Reticker-Flynn – David Wood – Yun Xue Acknowledgments SCBL – Joaquim Sampaio Cabral – Cláudia Lobato da Silva – Francisco Santos – Pedro Andrade S TEM C ELL B IOENGINEERING L ABORATORY