HyStem™ Cell Culture

Why Optimize ECM?

Matrix optimization is crucial
Each different cell type in a multicellular organism has its own unique microenvironment which is composed of several factors influencing the cell’s proliferative and differentiation status. In general, these factors fall into one of two categories: soluble cues (growth factors, metabolites, dissolved gases) and insoluble cues (the composition, architecture, and elasticity of the extracellular matrix (ECM) and cell-cell interactions)1,2. Since proliferation and differentiation of cells in culture has become increasingly important for basic research3, drug discovery4 and tissue engineering/regenerative medicine5, the incorporation of these cues for in vitro cultivation of cells is crucial for recreating a cell’s natural niche. Recently, emphasis has been placed on finding the appropriate mixture of the soluble cues in liquid media for culturing stem cells with minimal attention to the insoluble triggers 6-9. Insoluble cues such as the glycoproteins within the ECM however play significant roles in specifying cell fate by affecting the same signaling pathways used by soluble cues such as growth factors3. Recent data in mammalian cell and human mesenchymal stem cell differentiation show that the impact of insoluble cues; however, is far more profound than previously thought. In particular, the proper composition of the ECM, its presentation to the cell, and its appropriate stiffness significantly improve the biological relevance of an in vitro cell culture since the cell acts and looks far more like its in vivo counterpart in the presence of the appropriate soluble medium.
Matrix role in mammary epithelial cell differentiation
One example in mammalian cell culture that illustrates the utility of providing the appropriate ECM composition and presentation is in vitro mammary epithelial cell differentiation. Mammary epithelial cells have two requirements for in vitro differentiation into bilayered acini capable of producing milk proteins: the ECM must be properly presented to the cell and it should be laminin-rich. Mammary scp2 epithelial cells do not produce b-casein in the presence of prolactin when the cells are simply grown as two-dimensional monolayers; the monolayer must first be overlaid with a laminin-rich basement membrane extract10. Indeed, purified laminin by itself can substitute for the basement membrane extract, underscoring the additional importance of matrix composition10.
Matrix role in kidney ureteric bud morphogenesis
Another example in mammalian cell culture is branching morphogenesis during early kidney development. One of the first steps in early kidney development is the extensive branching morphogenesis of a structure known as the ureteric bud (UB) within surrounding mesenchymal tissue. To recapitulate UB branching in-vitro, three-dimensional culture with the appropriate soluble and insoluble cues plays a crucial role11. Simple hydrogels such as alginate-based gels and Puramatrix did not work, underscoring the need for a surrounding matrix fortified with additional ECM proteins and growth factors11.
Matrix stiffness helps determine a stem cell’s fate
Matrix stiffness, which matches that of a specific target cell’s in vivo microenvironment, acts as a potent signal to differentiate mesenchymal stem cells (MSCs) into the target cell. More importantly, recent data illustrates that specific matrix stiffness allows MSCs to more fully differentiate into the matching target cell type than it can with soluble inducers alone. In non-inductive media, matrix stiffness by itself is a strong inducer of MSC differentiation (measured in terms of morphology and tissue-specific gene and protein expression profiles) when MSCs are plated on matrices of different elasticities12. In tissue-specific inductive media; however, the levels of various tissue specific protein marker levels were comparable to in vivo levels only when the appropriate stiffness was matched to the correct inductive medium12. For example, in myogenic inductive media, the MyoD1 muscle marker protein was expressed at approximately half the level of that in C2C12 muscle cells when MSCs were plated on matrices of different stiffness. Only when the matrix stiffness matches that of muscle (11 kPa), are MyoD1 levels in differentiating MSCs and C2C12 cells comparable12.
Co-optimization of matrix and medium
In sum, the emerging picture for the future of cell culture is the imperative to optimize both the soluble medium and the insoluble cues. Since both sets of cues affect the same signaling pathways through crosstalk and the outcome of mixing these cues in one culture system cannot be predicted a priori 1,8,9,13, it stands to reason that both sets of cues must be co-optimized for best results. Co-optimization of the many combinations of both soluble and insoluble cues lends itself to high-throughput approaches where several groups have begun work 1,8,9,14,15. It is logical that such screening approaches will be crucial as ideal cell culture systems are developed for differentiating adult and embryonic stem cells for future applications in tissue engineering and regenerative medicine. In the future, since the purpose of cell culture optimization in vitro is to grow cells for in vivo therapies, much effort needs to be placed on developing hydrogels which can bridge both needs. The best hydrogel will not only be able to supplant well-performing, animal-based hydrogels such as Matrigel, but it will also act as an in vivo animal-free cellular delivery vehicle which is biocompatible, biodegradable, injectable, and flexible enough to allow its stiffness to match that of the target cell type for reasons described here.
References
  1. Flaim CJ, Chien S, Bhatia SN. An extracellular matrix microarray for probing cellular differentiation. Nat Methods. 2005 2:119-25.
  2. Scadden DT The stem-cell niche as an entity of action. Nature. 2006 41:1075-9.
  3. Hansen RK and Bissell MJ Tissue architecture and breast cancer: the role of extracellular matrix and steroid hormones. Endocr Relat Cancer. 2000 7:95-113
  4. McNeish J Embryonic stem cells in drug discovery. Nat Rev Drug Discov. 2004 3:70-80.
  5. Guillot PV, Cui W, Fisk NM, Polak DJ. Stem cell differentiation and expansion for clinical applications of tissue engineering. J Cell Mol Med. 2007 11:935-44.
  6. Ludwig TE, Bergendahl V, Levenstein ME, Yu J, Probasco MD, Thomson JA. Feeder-independent culture of human embryonic stem cells. Nat Methods. 2006 3:637-46.
  7. Lu J, Hou R, Booth CJ, Yang SH, Snyder M. Defined culture conditions of human embryonic stem cells. Proc Natl Acad Sci U S A. 2006 103:5688-93.M
  8. Soen Y, Mori A, Palmer TD, Brown PO. Exploring the regulation of human neural precursor cell differentiation using arrays of signaling microenvironments.Mol Syst Biol. 2006 2:37.
  9. Nakajima M, Ishimuro T, Kato K, Ko IK, Hirata I, Arima Y, Iwata H. Combinatorial protein display for the cell-based screening of biomaterials that direct neural stem cell differentiation. Biomaterials. 2007 28:1048-60.
  10. Roskelley CD, Desprez PY, Bissell MJ.Extracellular matrix-dependent tissue-specific gene expression in mammary epithelial cells requires both physical and biochemical signal transduction. Proc Natl Acad Sci U S A. 1994 91:12378-82.
  11. Rosines E, Sampogna RV, Johkura K, Vaughn DA, Choi Y, Sakurai H, Shah MM, Nigam SK. Staged in vitro reconstitution and implantation of engineered rat kidney tissue. Proc Natl Acad Sci U S A. 2007 104:20938-43.
  12. Engler AJ, Sen S, Sweeney HL, Discher DE.Matrix elasticity directs stem cell lineage specification. Cell. 2006 126:677-89.
  13. Schmeichel KL, Bissell MJ. Modeling tissue-specific signaling and organ function in three dimensions. J Cell Sci. 2003 116:2377-88.
  14. Underhill GH, Bhatia SN. High-throughput analysis of signals regulating stem cell fate and function. Curr Opin Chem Biol. 2007 11:357-66.
  15. Flaim CJ, Teng D, Chien S, Bhatia SN. Combinatorial signaling microenvironments for studying stem cell fate. Stem Cells and Development 2007 in press.