a Cell Biology Section, National Institute of Dental and Craniofacial Research, Bethesda, Maryland;( o1 X0 R0 w+ M
# H L+ Y9 y- j9 q. Db NASA/NIH Center for Three Dimensional Tissue Culture, National Institute of Child Health and Human Development, Bethesda, Maryland;$ D. S: R' m+ N! c* Q' L
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c George Washington University Medical Center, Washington, DC; % r" Z2 A6 S" E. [/ e, K2 B0 b' q7 f+ z8 `
d Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Human Development, Bethesda, Maryland, USA " z5 O, ^# x. V, I: r9 I/ j6 z+ A" c: M" V' t0 Z
Key Words. Matrigel ? Basement membrane ? Extracellular matrix ? Gland formation ? Chondrogenesis2 f) Y- w( t5 w+ {& o: O6 w
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Correspondence: Leonid Margolis, Ph.D., NIH, NICHD, Building 10, Room 10D58, Bethesda, MD 20892 USA. Telephone: 301-594-2476; Fax: 301-480-0857; e-mail: margolis@helix.nih.gov& P) z: a, \9 j; C+ a& g3 q
9 @4 s5 Q) {- d: |) L5 jABSTRACT 6 Q: I9 a% T2 c0 m$ a `! J. x* ?: z# F0 x7 mThe extracellular matrices in tissues are likely to be the first molecular components with which stem cells interact. Stem cell niches are thought to be important for programming their differentiation. These complex extracellular matrices function in tissues to provide support and store growth factors and cytokines as well as biological signals to promote and maintain cell differentiation . The effect of individual matrix components as well as complex matrices on differentiation of various cells has been well documented using in vitro assays, but their effect on stem cell differentiation is less well studied. The basement membrane matrix, which underlies epithelial and endothelial cells and contacts smooth muscle, fat, and peripheral nerve cells, is highly enriched in growth factors and has been shown to promote the differentiation and formation of tissue-like structures of many cell types. In particular, endothelial cells attached to a complex mixture of basement membrane components, termed Matrigel or EHS gel , form capillary-like structures with a lumen . Likewise, freshly isolated Sertoli cells form chord-like structures, and salivary gland cells form acinar-like structures on Matrigel . The cells on basement membrane matrices are more differentiated based on the activation of cell type–specific genes . For example, breast epithelial cells form glandular-like structures and produce casein when cultured on basement membrane matrix . In addition, salivary gland cells produce amylase when cultured on basement membrane matrix but not when cultured on either collagen I or laminin-1, biologically active components of skin and the basement membrane, respectively . 0 n, s4 w- y& r& R $ w* j* z$ @, z5 H- l( _Although much has been studied with basement membrane and its components, less has been accomplished with other extracellular matrices. It would be expected that such matrices also would have cell type–specific effects on cell differentiation. Recently, we developed a new matrix, an extract of cartilage, termed Cartrigel, using the same methodology with minor modifications as was used for the basement membrane . In preliminary studies, this material was found to sustain chondrocyte differentiation.6 }3 B5 C: F4 E( D9 a/ q1 @' e( `
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In this study, we have examined the biological effects of basement membrane (Matrigel) and of cartilage (Cartrigel) extracts on the differentiation of pluripotent rhesus monkey embryonic stem (ES) cells in conventional monolayer culture and in a rotating wall vessel (RWV) bioreactor, which was shown to facilitate differentiation of various cells. In this NASA-designed device, cells grow in suspension, form large aggregates, and are subjected to minimal shear forces . We find that Matrigel can promote rapid and specialized cell differentiation with more differentiated cells in the RWV bioreactor than in monolayer cultures, whereas some of the individual components of Matrigel cannot stimulate differentiation in either culture condition. Basement membrane extra-cellular matrix (Matrigel) promoted immature glandular/epithelial differentiation, whereas cartilage extract (Cartrigel) promoted the formation of mature and calcified cartilage. * T( N# o8 Q7 y' m- C ) o: j$ L1 g. F4 ]6 nMATERIALS AND METHODS + Z. J3 I, x5 }; ]5 S( q7 y# r% d6 x
We first examined the effects of various extracellular matrices on the differentiation of rhesus monkey ES cells in vitro (Table 1). Based on analysis of the morphology, the basement membrane component laminin-1 and collagen I had little effect on cell survival or differentiation when added as a substrate in place of the feeder layer or as a soluble reagent in the presence of the feeder layer (Table 1). The basement membrane extract Matrigel, either in solution or as a substrate in the absence of the feeder layer, induced cell growth and differentiation. By 4 days in culture, several different cellular morphologies were observed in the same well. In addition to isolated single cells, large tubular-like structures were observed along with areas of cell aggregates (Fig. 1B), whereas the control (no Matrigel or Cartrigel) cells alone formed a monolayer (Fig. 1A). By day11 in culture on Matrigel, the cells had continued to form large organ-like tubular structures as well as highly condensed areas in which a clear morphology could not be determined (Figs. 1C, 1D). Based on morphological comparisons with control cell cultures, it was apparent that proliferation was increased in the presence of Matrigel and that the cells were not just migrating into clumps. Individual cells and structures within the clumps could not be easily observed with light microscopy of living samples. Therefore, we fixed, sectioned, and stained cells grown on Matrigel. H&E staining revealed that many glandular-like structures could be observed with the cells grown on Matrigel. Some contained polarized columnar epithelial-like cells without a lumen (Fig. 1G), whereas others had lumens of varying sizes (Figs. 1E, 1F, 1H). S2 x; r! X! [" O! @8 v
. Q6 R$ L: w4 r v+ J: m, J: v: G; y7 fTable 1. Effect of various extracellular matrix components on the differentiation of monkey embryonic stem cells in vitro and growth in vivo: w' w- O7 I( r" ?% u/ [
6 ~4 _0 x u& u' `, q1 xFigure 1. In vitro culture of rhesus monkey embryonic stem cells alone (A) and on Matrigel substrates (B–H). (A): Embryonic stem cells on murine embryonic fibroblasts after 11 days. (B): Cells on Matrigel after 4 days. (C, D): Cells on Matrigel after 11 days. (E–H): Histological section of cells stained with hematoxylin and eosin after 11 days of culture on Matrigel.! q' f6 S1 w( v9 v/ f' J2 m" J
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Cells were also grown in RWV bioreactors, and the effects of laminin-1 and basement membrane Matrigel were assessed. In RWV bioreactors, the three-dimensional in vivo environment may be reflected more faithfully. With laminin-1, cells formed few polarized structures (Figs. 2A–C), similar to control cultures. A small structure shown in Figures 2A–C was highly infrequent and shows poor cellular organization. In contrast, addition of soluble Matrigel into the RWV cultures clearly increased the size of the cell aggregates with time of culture (data not shown), and the number of highly organized tubular- and glandular-like structures was much greater than that observed in the control. In addition, some areas appeared to contain muscle-like cells (Fig. 3F). There were also many more tubular- and glandular-like structures in the RWV bioreactor culture (approximately 20%–40%) (Figs. 2A–C) than in the cultures from the dishes in the presence of Matrigel (approximately 10%–20%). Staining with laminin antibodies confirmed the polarized nature of the cell clusters even when the cells did not appear polarized (Fig. 2D). We immunostained the cells in the RWV and in the tumors with markers for epithelial cells (cytokeratin, AE1/ AE3), neuronal cells (class III ? tubulin), and mesenchymal cells (V9). The tumor-derived polarized tubular- and glandular-like cells stained with the cytokeratin antibody, which is a marker of epithelial cells (Figs. 3A, 3B). Approximately 30% of the cells, entirely in the nonglandular areas, stained with the marker for mesenchymal cells (Figs. 3E, 3F), whereas clusters of cells were positive with the neuronal markers (Figs. 3C, 3D) only in the RWV cultures, with no neuronal cells observed in the tumors. No cells were positive for the endothelial cell lineage (data not shown). Thus, the cells in the RWV and in the tumors were of mixed cell types, with the dominant structures being epithelial./ [1 B" [4 T- v8 k+ |$ d' e
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Figure 2. (A–C): RWV culture of rhesus monkey embryonic stem cells alone (A) and in the presence of Matrigel (B, C) for 30 days. (A): Cells alone. (B, C): Cells cultured with soluble Matrigel. All sections were stained with hematoxylin and eosin. (D): RWV culture of rhesus monkey stem cells in the presence of Matrigel and stained for laminin-1. Abbreviation: RWV, rotating wall vessel. % Y0 |3 k) l8 e$ @( r6 h- l1 l& d3 T; j$ Q( u4 l: ^
Figure 3. Immunostaining of cells grown in the RVW and in vivo in the presence of basement membrane Matrigel. (A, B): Immunostaining for cytokeratin in cells in RVW (A) and in tumors (B). (C, D): Immunostaining for neuronal cells in cells in RVW. (E, F): Immunostaining for vimentin in cells in RVW (E) and in tumors (F). Abbreviation: RWV, rotating wall vessel. 9 u4 W; o" ~8 S8 B# B) I9 d7 b 2 c' \9 B5 V& H2 ?, k9 o. z- |The nature of these structures was confirmed by electron microscopy, in which they were found to appear as immature glands (Fig. 4). The untreated control cells grew predominantly as flat monolayers joined to one another at their free surface by terminal bars comprised of a complex of tight and intermediate junctions and desmosomes (Figs. 4A). Pleomorphic villous processes lacking core rootlets or a glycocalyx sparsely covered the free surface. Some areas of the basal surface were coated by a basal lamina. The cytoplasm contained small amounts of intermediate filaments, consistent with vimentin, as well as thin actin filaments. Cellular debris was seen in the cytoplasm of some cells, consistent with apoptosis. " n3 M. Q& o- a9 G- M) c+ d7 o4 M% {9 \' g9 V
Figure 4. Electron microscopy of rhesus monkey embryonic stem cells alone (A) and in the presence of Matrigel (B, C) for 21 days in culture. (A): Control. The apical surface of two control cells is joined by a well-developed terminal bar. Note the presence of several microvilli and a coated pinocytotic pit. (B): Matrigel, low magnification. This tubular structure is comprised of several layers of cells that are partially separated by gaps. A single cell is in the process of dividing. (C): Matrigel, higher magnification of (B). The luminal surface of the cells has pleomorphic microvilli and junctional complexes. The cells bulge into the lumen.+ F, y* e5 o" M; w1 r- a# l( U( m
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Cells grown on Matrigel mainly differed from the control cell cultures by their unique organization: aggregates of varying sizes and tubular structures. The tubular structures varied from having a single layer of cells to having multiple layers. Rare desmosomes joined lateral surfaces of the cells. Most cells were round to oval in shape, with occasional columnar cells. These cells also had terminal bars, pleomorphic microvilli, and stretches of basement membrane (Figs. 4B, 4C).8 I6 g/ d5 q6 _$ W- ?: S! N8 `( b
" @1 V2 c: h" V r# wNext, we studied further differentiation of these cells in the presence and absence of laminin-1 and Matrigel upon intramuscular and subcutaneous injection. In 5 months, we did not observe in vivo growth of cells cultured in RWV or on monolayers with laminin-1. With Matrigel-injected cells, tumors did grow as previously shown . 9 j% q2 B1 V5 W$ F# K9 V % i9 F9 u( A i1 }8 M) }: \In addition, we determined the biological effect of another complex extracellular matrix, Cartrigel, which was prepared from cartilage in a manner analogous to the procedure used for Matrigel. In monolayer, the cells were cultured in vitro in the presence of a Cartrigel substrate or, when it was present in solution, seemed to increase in number with some condensation, but clear structures could not be observed even after H&E histological staining (data not shown). Similarly, in the RWV bioreactor, the cell number increased several-fold, with the aggregates of cells some 5- to 10-fold larger than controls, but no obvious differentiation was observable (data not shown). When these cells were injected subcutaneously in mice after incubation in the RWV bioreactor for 21 days, growing tumors in 50% of the mice injected were observed at the injection site within 2 months. When examined by histology, large cartilage nodules were found that stained with alcian blue (Figs. 5A, 5C, 5D). Some of the cartilage nodules appeared calcified, as demonstrated by von Kossa staining (Fig. 5B). When Cartrigel was injected alone, no tumors were observed, and the material seemed to be absorbed, because it could not be found in the tissue. This study demonstrates that complex extracellular matrices can drive cell differentiation along different pathways depending on the source of the matrix components. y0 e2 D3 E3 S. E+ e; ^
5 O; V' h6 D; p1 O& ^Figure 5. Histology of tumors formed after 15 weeks of injection of embryonic stem cells into mice. Embryonic stem cells were first cultured in vitro with Cartrigel and then injected. (A): Hematoxylin and eosin section of tumor tissue. (B): Von Kossa stain of tumor tissue. (C, D): Alcian blue stain of tumor tissue. ' e0 p O0 q0 e( r7 n& `, [. q2 U* m& q, r X7 W; }/ B
DISCUSSION2 t' J, K4 ]9 [ F- I
$ m3 |$ n6 ]4 W0 I. O6 C% nThis work was supported by intramurals funds of National Institute of Dental and Craniofacial Research and National Institute of Child Health and Human Development and by the NASA-NIH Center for Tissue Culture.' n) {! H$ G& z
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