Glycosan offers three options for Stem Cell Culture:
- HyStem: for applications requiring attachment factor optimization. Extracellular matrix proteins can be mixed into the hydrogel and incorporated non-covalently before gelation. Alternatively, attachment peptides having an N-terminal cysteine can also be covalently linked to the matrix.
- HyStem-C®: a general starting point for optimization of a cell’s microenvironment. HyStem-C contains Gelin-S or thiolated gelatin (denatured collagen) which allows attachment of a wide variety of cell types and takes the guesswork out of the appropriate attachment factors to use.
- HyStem-HP®: for applications requiring slow release of growth factors in a cell’s microenvironment. HyStem-HP contains small amounts of thiolated heparin which ionically binds a wide variety of growth factors and slowly releases them over time17,18,19.
“HA hydrogels act as a unique microenvironment for the propagation of human embryonic stem cells (hESCs), likely due to the regulatory role of HA in the maintenance of hESCs in the undifferentiated state, in vitro as well as in vivo.”3
HyStem® hydrogels are designed for stem cell culture. They are chemically defined and wholly animal free. They are based on hyaluronan (HA) – a component of the extracellular matrix (ECM) that is abundant in embryos and stem cell niches. HyStem hydrogels provide a compliant, viscoelastic matrix that is physiologically relevant for stem cells. They emulate a stripped down version of the ECM to which additional components (such as growth factors and ECM proteins) can be added to recreate the composition of a specific niche.
Hyaluronan relevancy for stem cells
HyStem hydrogels are composed of HyStem (thiol-modified hyaluronan, HA) and Extralink® (thiol-reactive crosslinking agent). HA is the simplest glycosaminoglycan (a class of negatively charged polysaccharides) and a major constituent of the ECM1,2. Embryonic ECM possesses a high quantity of glycosaminoglycans of which HA is predominant3. Human embryonic stem cells (H1, H9 and H13 lines) have been shown to express both CD44 and CD168 (RHAMM), which are HA receptors. Human embryonic stem cells (hESCs) have also been cultivated by encapsulating them in HA hydrogels. These cells were grown for 15 days without detectable differentiation. After recovery from the hydrogels, the hESCs could be differentiated using endothelial growth media supplemented with VEGF3.
HA is also present in large amounts in the ECM of embryonic livers, fetal livers and the putative stem cell niche within the liver (the Canals of Hering). Hepatic stem and progenitors cells (hepatoblasts) have been successfully cultured for over 4 weeks without differentiation when encapsulated in HA hydrogels and grown with a defined media (Kubota’s medium). Additionally, these hepatic progenitor cells express CD44 at high levels4.
Defined and animal-free hydrogel
When used with defined media, HyStem allows for the culture of stem cells in a defined system. The HA used to produce HyStem is made by a proprietary bacterial fermentation process using the recombinant hasA gene fromStreptococcus equisimilis, which is expressed in Bacillus subtilis5, as the host in an ISO 9001:2000 process. It is 100% free of animal-derived raw materials and no animal derived ingredients are used in its production. The polyethylene glycol diacrylate that is our Extralink is an acrylate modification of polyethylene glycol (PEG), which is a commodity chemical. PEG is derived from petroleum and inorganic sources and contains no animal source materials.
Stem cells can be encapsulated in HyStem prior to crosslinking7, where they grow within the hydrogel matrix. Cells are removed from the hydrogel by digesting it using hyaluronidase.
If ECM proteins are added to HyStem prior to crosslinking they are non-covalently incorporated into the matrix . If the relevant protein is added, the stem cells can be plated on top of the hydrogel for pseudo three dimensional growth1. Since the researcher determines which ECM proteins to incorporate, the sourcing and concentration of the proteins is fully under their control. It may be optimal to begin experimentation using animal derived ECMs that are compatible with humans, but less expensive. However, the method of purification can affect its performance as can the specific isoform of the ECM protein. Therefore, results achieved with animal proteins will not necessarily be mirrored in their commercially available human counterparts.
Additionally, once the relevant ECM proteins are determined for your cell type, we recommend doing a concentration variation study to determine the minimum protein amount required for your application. Cells are passaged using dispase or collagenase.
HyStem hydrogel variation
ECM protein incorporation
Stem cells expand when encapsulated in HA only hydrogels. However, of the cells tested to date, none attach to HyStem hydrogels1,2. For most applications, optimal cell proliferation will require attachment. This can be achieved by incorporating specific ECM proteins into the HyStem® hydrogels prior to crosslinking. Since the appropriate ECM protein depends upon the cell type and the desired outcome (proliferation without differentiation vs differentiation), HyStem® gives the user the flexibility to decide on the appropriate experimental conditions and material sources (human vs animal).
During embryogenesis, laminin is the first ECM protein to be excreted within the basement membrane. It is observed in a punctuate pattern in the intercellular spaces between cells in the 8 cell stage embryo. Later in development fibronectin, heparan sulfate and collagen IV accumulate in the same area. The deposition and self-assembly of collagen IV leads to the organization of the basement membrane and hence to the organization of the attached cells, leading to the polarization of the epithelial monolayer8.
The rigidity of HyStem hydrogels can easily be adjusted. Depending upon your application, the hydrogel stiffness has been found to be very important in stem cell cultures9,10. The standard HyStem hydrogel made per our instructions has a compliance of ~300 Pa11. The hydrogel compliance can also be altered either by varying the amount of crosslinker or by diluting the hydrogel solutions.
Growth factor incorporation
HyStem may be used to conserve growth factors (GFs) while extending their active life. For media that use GFs, it is possible to move the GFs from the media to the HyStem® hydrogel. GFs are retained within and slowly released from the HyStem-based hydrogels over several weeks12,13. They are protected from proteolysis so that their bioactivity is maintained12,13. To date we have characterized release of 7 GFs (bFGF, VEGF, KGF, PDGF, TGF-β1, Ang-1, HGF)12,13,14. Experimentation will be required to determine the optimal GF concentration in the hydrogel for your application.
Stem cells cultured
The following stem cells have been cultured in HyStem-based hydrogels:
- human embryonic stem cells (H9)7
- human cord blood derived CD34+ stem cells7
- human mesenchymal stem cells7
- human hepatic progenitor cells4
- rabbit mesenchymal stem cells15
- X.Z. Shu, S. Ahmad, Y. Liu, and G.D. Prestwich, “Synthesis and Evaluation of Injectable, in situ Crosslinkable Synthetic Extracellular Matrices (sECMs) for Tissue Engineering,” J. Biomed Mater. Res. A, 79A(4), 901-912 (2006).
- X.Z. Shu, Y. Liu, F. Palumbo, G.D. Prestwich, “Disulfide-crosslinked Hyaluronan-Gelatin Hydrogel Films: A Covalent Mimic of the Extracellular Matrix for In Vitro Cell Growth,” Biomaterials, 24, 3825-3834 (2003).
- S. Gerecht, J.A. Brudick, L.S. Ferreira, S.A. Townsend, R. Langer, G. Vunjak-Novakovic, “Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells,” PNAs 104(27): 11298-11303.
- Unpublished data from W. Turner, R. Turner, L. Reid, University of North Carolina (publications submitted August 2007).
- B.Widner, R. Behr, S. Von Dollen, M. Tang, T. Heu, A. Sloma, D. Sternberg, P.L. DeAngelis, P.H. Weigel, S. Brown, “Hyaluronic Acid Production in Bacillus subtilis”, Applied and Environmental Microbiology 71(7): 3747-3752 (2005).
- S. Cai, Y. Liu, X.Z.Shu, G.D.Prestwich, “Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor,”Biomaterials, 26, 6054-6067 (2005).
- Unpublished data from T. Tandenski, L. Kelley, University of Utah.
- Ingber, D. “Mechanical control of tissue morphogenesis during embryological development,” Int. J. Dev. Biol. 50: 255-266 (2006).
- A.J. Engler. S. Sen. H.L. Sweeney, D.E. Discher, ”Matrix Elasticity Directs Stem Cell Lineage Specification,” Cell, 126, 677-689 (2006).
- T. Yeung, P.C. Georges, L.A. Flanagan, B. Marg, M. Ortiz, M. Funaki, N. Zahir, W. Ming, V. Weaver, P.A. Jamney, “Effects of Substrate Stiffness on Cell Morphology, Cytoskeletal Structure, and Adhesion, “Cell Motility and the Cytoskelton“, 60, 24-34 (2005).
- Unpublished data from Janssen Vanderhooft, Glenn Prestwich, University of Utah.
- D. B. Pike, S. Cai, K.R. Pomraning, M.A. Firpo, R.J. Fisher, X.Z. Shu, G.D. Prestwich, R.A. Peattie, “Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF”, Biomaterials, 27, 5242–5251 (2006).
- Unpublished data from Rob Peattie lab (Oregon State University) and S. Cai and B. Yu of Glenn Prestwich (University of Utah) lab.
- Unpublished data from Yongzhi Qiu, Robert McCall, Vladimir Mironov, Xuejun Wen, Clemson University and Medical University of South Carolina.
- Y. Liu, X.Z. Shu, G. D. Prestwich, “Osteochondral defect repair with autologous bone marrow derived MSC cells in an injectable in situ crosslinked synthetic extracellular matrix” Tissue Engineering, Tissue Eng., 12(12), 3405-3416 (2006).
- X. Z. Shu, K. Ghosh, Y. Liu, F. S. Palumbo, Y. Luo, R. A. Clark, and G. D. Prestwich, “Attachment and spreading of fibroblasts on an RGD peptide-modified iInjectable hyaluronan hydrogel” J. Biomed. Mat. Res. 68A, 365-375 (2004).
- S. Cai, Y. Liu, X.Z. Shu, G.D. Prestwich, “Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor”,Biomaterials, 26, 6054-6067 (2005).
- D. B. Pike, S. Cai, K.R. Pomraning, M. A. Firpo, R. J. Fisher, X. Z. Shu, G. D. Prestwich, R. A. Peattie, “Heparin-regulated release of growth factors in vitro and angiogenic response in vivo to implanted hyaluronan hydrogels containing VEGF and bFGF”, Biomaterials, 27, 5242-5251 (2006).
- Unpublished data from G. D. Prestwich, et al, University of Utah, and R. Peattie, et al, University of Oregon.