Original Paper International Journal of Cell Cloning 8:425-430 (1990)
Hematopoiesison CelluloseEster Membranes (CEM). XU. Effect of Membrane Enrichment by Purified Matrix Proteins William H . Knospe, Salah G. Husseini, David Schwartz Section of Hematology, Departments of Medicine and Biochemistry, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois, USA
Keys Words. Hematopoiesis
Stromal cells
- Matrix proteins
Abstract. Cellulose ester membranes (CEM) were enriched with the following puri-
fied matrix proteins: collagen I, II, IV,proteoglycan and laminin. Fifteen milligrams of each were placed on CEM which were then folded into open-ended tubes implanted i.p. and S.C. CEM were removed after 3,6 and 12 months and examined histologically. There was no evidence of hematopoiesis or new bone formation on the implanted, enriched CEM at any of the intervals examined. Collagen I and proteoglycan-enrichedCEM showed evidence of increased sinusoid-like vascular structures.
Introduction Studies of hematopoiesis on cellulose ester membranes (CEM) indicated that hematopoietic stromal cells from marrow or bone also secreted noncellular matrix factors, including glycosaminoglycans (GAGS),proteoglycans (PGs) and glycoproteins [l, 21. Enrichment of CEM by the same types of stromal cells resulted in the formation of new bone, and the processes of osteogenesisand hematopoiesis were closely linked. The secretion of sulfated, non-acidic glycoproteinscorrelated best with the processes of hematopoietic and osteogenic regeneration on the stromal cell-enriched CEM [2]. GAGs and PGs modulate and stimulate granulopoiesis in long-term marrow cultures supported by strornal cell layers. Zuckerman and Wcha identified a variety of matrix proteins in the adherent layers of long-term marrow cultures, including several collagen species (including collagen IV and laminin) and related these matrix proteins to endothelial cells [3-51. Correspondence:William H.Knospe, M.D., Director, Section of Hematology, RushPresbyterian-St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612,USA. Received May 14, 1990; accepted for publication July 6, 1990. 0737-1454/90/$2.00/0 oAlphaMed Press
Knospe/Husseini/Schwartz
426
The present studies were undertaken to determine if purified matrix proteins and proteoglycans placed on CEM could facilitate the development of a stroma capable of supporting hematopoiesis.
Materials and Methods CAF, female mice (Cumberland View Farms, Clinton, TN), 12-14 weeks of age, were used in all experiments. Coated and uncoated CEM 1.5 X 1.0 cm were folded into thirds to form an open-ended trilaminar structure (Millipore Corp., Bedford, MA). Mice were anesthetized with 1.4 mg of Nembutal i.v. The abdomen was opened with a midline incision, and CEM coated with matrix components were implanted peritoneally between the mesentery and the abdominal wall. Other CEM coated with matrix components were implanted into thoracic S.C. sites. Five to 7 mice were killed after intervals of 3,6 and 12 months. The CEM were removed, cleaned of adherent tissue, fixed in neutral formalin and embedded in paraffin. Sections were cut at 4-6 pm, stained with hemotoxylin and eosin, and examined microscopically for the presence of bone and hematopoiesis. At each point studied, 6 to 14 implants were examined at i.p. and S.C. implantation sites. Coating of CEM Uncoated negative control CEM were implanted into the peritoneal cavity and S.C. sites without any prior treatment. Positive control CEM were coated on one side with a thick cellular suspension of bone marrow extruded from donor mouse femurs by cutting off the epiphyses and flushing the medullary cavities with 1.Oml of Hank's solution (GIBCO, Grand Island, NY) injected into the medullary cavity through a no. 21 needle under pressure. Bone homogenate was prepared by removing 1 femur from donor mice, clipping off the epiphyseal ends, flushing the medullary cavity free of all marrow, splitting the diaphysis into halves and gently grinding them in a sterile mortar and pestle with 1.0 ml of Hank's solution. The paste obtained from one-half of a femur was then spread on one side of a CEM . Purified matrix components were similarly applied and spread uniformly Over one surface of a CEM. Fifteen-milligram quantities of each species were placed on a single CEM. Combination of collagen I and PG (15 mg quantities of each) were also placed on CEM and implanted i.p. and S.C. Prepamtion of Purified Mat& Proteins and PG
Type I Collagen from Murine Bone Femurs were decalcified by repeated changes of 1.0 M EDTA solutions at 4°C over 5 days. Bone residue (now C a m , free) was resuspended in 0.5 M acetic acid containing 1.0 mglml pepsin and digested at 4°C for 24 h. Supernatant (containing collagen) was precipitated by the addition of NaCl to a 5% final concentration. Precipitate was redissolved in 0.5 M acetic acid and reprecipitated with NaCl as above. Precipitate was washed with phosphate-buffered saline (PBS). Murine Cartilage Collagen flype II) and Proteoglycan Prepamtion Mouse sternae were suspended in 4.0 M guanidine chloride (Sigma Chemical Co., St. Louis, MO) and 0.05 M Tris, pH 7.4, for 24 h. Solubilized material (containing proteoglycans) and insoluble residue (containing type II collagen) were processed separately.
Matrix Proteins and Hematopoiesis
427
Collagen Residue was acidified with acetic acid and digested with 1.0 mg/ml pepsin (Sigma) at 4°C overnight. The residue was then neutralized and NaCl added to 3.5 M. This resulted in the solubilization of type II collagen. The soluble material was then reprecipitated and washed in PBS. Proteoglycans The 4 M guanidine chloride extract was mixed with 2 times its volume of cold 100% ethanol. The precipitate was collected and redissolved in PBS. Any remaining precipitate was diswded. The supernatant was precipitated as purified protmglycan with 100%ethanol.
Laminin Semipurifiedlaminin in the form of Matngel (Collaborative Research, Bedford, MA) was used in these experiments. This material is 70% laminin, 15% collagen IV, 10% heparin sulfate and 5% proteoglycan and eutactine. Collagen IV (Bovine Lens Protein) Bovine lens capsule was solubilized in 0.5 M acetic acid at 4U'C. The solubilized material was precipitated with 0.5 N NaCl which was then dialyzed to neutral. The precipitated material was centrifuged and resuspended in neutral phosphate buffer.
Results None of the CEM enriched with purified noncellular matrix species implanted either i.p. or S.C. developed bone or hematopoiesis. One unexpected observation was that CEM with collagen I and proteoglycan developed extensive sinusoid-like structures, but there was no hematopoiesis associated with these vessels (Fig. 1). Combinations of collagen I and proteoglycan did not enhance the development of sinusoids or induce bone or hematopoiesis. The CEM coated with purified matrix species became increasingly fibrotic with extensive collagen deposition. The negative control uncoated CEM implanted i.p. and S.C. were also devoid of any bone formation or hematopoiesis. These CEM became increasingly fibrotic compared with CEM with non-cellular matrix proteins and PGs. The positive controls coated with living stromal cells from bone or bone marrow developed extensive trilineal hematopoiesis and new bone formation after 6 and 12 months i.p. implantation [l].
Discussion These experiments were negative in the sense that they did not provide evidence for an inductive or modulatory role for hematopoiesis or bone formation for any of the matrix species studied. However, they do not exclude a role for other matrix factors such as the bone-inducing glycoprotein osteogenin present in bone and tooth matrix powder [6-81. We have demonstrated that stromal cells from bone or bone marrow secrete
Knospe/Husseini/Schwartz
428
Fig. 1. CEM coated with proteoglycanand implanted for 6 months i.p. Hematoxylin . is extensive sinusoid and eosin stain. Original magnification(A) 4x and (B) 4 0 ~There formation throughout the interior of the CEM (arrows). Hematopoiesisand bone are absent.
Matrix Proteins and Hematopoiesis
429
a similar factor which is adsorbed onto the enriched CEM after intervals of 1-6 months residence in the peritoneal cavity. Subcutaneously implanted CEM containing only non-cellular matrix factors rapidly induced new bone and hematopoiesis, but similar i.p. implanted CEM did not develop new bone or hematopoiesis [9, 101. Recently, we studied GAG and PG species in S.C. implanted tooth matrix and bone matrix. Initially, the implants contained abundant GAGs which appeared to be involved in the attraction of protomesenchymal cells resident only in skin and not in other tissues, including peritoneal cavity. These cells were transformed by osteogenin to become bone-forming cells and hematopoietic stromal cells. Later, the transformed bone cells formed proteoglycan species. The inactive bone matrix implants did not contain significant amounts of GAGs or PGs [lo-121. It is probable that PGs may modulate hematopoiesis, and yet not affect stromal cells [3-51. The observation that collagen I and proteoglycan seem to stimulate capillary or sinusoid-like development is interesting. Vascular cells alone may be ineffective in forming a hematopoietic microenvironment, but in concert with other stromal cells and noncellular matrix may be able to form a hematopoietic microenvironment [13, 141.
Acknowledgment This work was supported by a contract from the U.S. Office of Naval Research (contract number N00014-84-(2-0302).
References Knospe WH, Husseini SG, Adler SS. Hematopoiesis on cellulose ester membranes (CEM). In. Long-term histologic changes between 6 and 12 months after i.p. implantation. Exp Hematol 1983;ll:Sl2-521. McCuskey RS, Meineke HA, Pinkstaff CA,Knospe WH. Hematopoiesis on cellulose ester membranes (CEM). VI. Histochemical evaluation of stromal-CEM interactions after stromal enrichment. Exp Hematol 1984;12:25-30. Zuckerman KS,Wicha MS. Extracellular matrix production by the adherent cells of long-term munne bone marrow cultures. Blood 1983;61:540-547. Spooncer E, Gallagher JT, Krizsa F, Dexter TM. Regulation of haemopoiesis in longterm bone mamw cultures. IV.Glycosaminoglycan synthesis and the stimulation of haemopoiesis by 0-D-xylosides. J Cell Biol 1983;96:510-514. McCuskey PA, McCuskey RS,Meineke HA. Studies of the hemopoietic microenvironment. IV. In v i m microscopic and histochemical study of allograftsof bone marrow in the hamster cheek pouch. Exp Hematol 1975;3:297-308. Reddi AH, Huggins CB. Formation of bone marrow in fibroblast-transformation ossicles. Proc Natl Acad Sci USA 1975;72:2212-2216. Reddi AH. Collagen and cell differentation. In: Ramachandran GN, Reddi AH, eds. Biochemistry of Collagen. New York: Plenum Publishing Corp., 1976:449-478. Sampath TK,Muthukumaran N, Reddi AH. Isolation of osteogenin, an extracellular matrix-associated, bone inductiveprotein, by heparin affinity chromatography. Proc Natl Acad Sci USA 1987;84:7109-7ll3.
KnospeIHusseinilSchwartz
430
9 Knospe WH, Husseini SG. Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM. Exp Hematol 1986;14975-980. 10 Knospe WH, Husseini SG, Fried W. Hematopoiesis on cellulose ester membranes. XI. Induction of new bone and a hematopoietic microenvironment by matrix factors secreted by marrow stromal cells. Blood 1989;74:66-70. 11 Knospe WH, Husseini SG, Adler SS, Reddi AH. Decalcified tooth matrix powder induces new bone formation and hematopoietic microenvironment in the mouse. Int J Cell Cloning 1985;3:320-329. 12 McCuskey RS, Reddi AH, Knospe WH. Histochemical evaluation of the hemopoietic microenvironment induced by subcutaneous implants of powdered tooth matrix or bone in mice. Blood 1985;66(suppl 1):l32a. 13 Knospe WH, Blom J, Crosby WH. Regeneration of locally irradiated bone marrow. I. Dose dependent, long-term changes in the rat, with particular emphasis upon vascular and stromal reaction. Blood 1966;28:398-415. 14 Knospe WH, Blom J, Crosby WH. Aplastic anemia. Dependence of function on structure in the bone marrow. Blood 1967;30:851.
Hematopoiesison CelluloseEster Membranes (CEM). XU. Effect of Membrane Enrichment by Purified Matrix Proteins William H . Knospe, Salah G. Husseini, David Schwartz Section of Hematology, Departments of Medicine and Biochemistry, Rush-Presbyterian-St. Luke’s Medical Center, Chicago, Illinois, USA
Keys Words. Hematopoiesis
Stromal cells
- Matrix proteins
Abstract. Cellulose ester membranes (CEM) were enriched with the following puri-
fied matrix proteins: collagen I, II, IV,proteoglycan and laminin. Fifteen milligrams of each were placed on CEM which were then folded into open-ended tubes implanted i.p. and S.C. CEM were removed after 3,6 and 12 months and examined histologically. There was no evidence of hematopoiesis or new bone formation on the implanted, enriched CEM at any of the intervals examined. Collagen I and proteoglycan-enrichedCEM showed evidence of increased sinusoid-like vascular structures.
Introduction Studies of hematopoiesis on cellulose ester membranes (CEM) indicated that hematopoietic stromal cells from marrow or bone also secreted noncellular matrix factors, including glycosaminoglycans (GAGS),proteoglycans (PGs) and glycoproteins [l, 21. Enrichment of CEM by the same types of stromal cells resulted in the formation of new bone, and the processes of osteogenesisand hematopoiesis were closely linked. The secretion of sulfated, non-acidic glycoproteinscorrelated best with the processes of hematopoietic and osteogenic regeneration on the stromal cell-enriched CEM [2]. GAGs and PGs modulate and stimulate granulopoiesis in long-term marrow cultures supported by strornal cell layers. Zuckerman and Wcha identified a variety of matrix proteins in the adherent layers of long-term marrow cultures, including several collagen species (including collagen IV and laminin) and related these matrix proteins to endothelial cells [3-51. Correspondence:William H.Knospe, M.D., Director, Section of Hematology, RushPresbyterian-St. Luke’s Medical Center, 1653 West Congress Parkway, Chicago, IL 60612,USA. Received May 14, 1990; accepted for publication July 6, 1990. 0737-1454/90/$2.00/0 oAlphaMed Press
Knospe/Husseini/Schwartz
426
The present studies were undertaken to determine if purified matrix proteins and proteoglycans placed on CEM could facilitate the development of a stroma capable of supporting hematopoiesis.
Materials and Methods CAF, female mice (Cumberland View Farms, Clinton, TN), 12-14 weeks of age, were used in all experiments. Coated and uncoated CEM 1.5 X 1.0 cm were folded into thirds to form an open-ended trilaminar structure (Millipore Corp., Bedford, MA). Mice were anesthetized with 1.4 mg of Nembutal i.v. The abdomen was opened with a midline incision, and CEM coated with matrix components were implanted peritoneally between the mesentery and the abdominal wall. Other CEM coated with matrix components were implanted into thoracic S.C. sites. Five to 7 mice were killed after intervals of 3,6 and 12 months. The CEM were removed, cleaned of adherent tissue, fixed in neutral formalin and embedded in paraffin. Sections were cut at 4-6 pm, stained with hemotoxylin and eosin, and examined microscopically for the presence of bone and hematopoiesis. At each point studied, 6 to 14 implants were examined at i.p. and S.C. implantation sites. Coating of CEM Uncoated negative control CEM were implanted into the peritoneal cavity and S.C. sites without any prior treatment. Positive control CEM were coated on one side with a thick cellular suspension of bone marrow extruded from donor mouse femurs by cutting off the epiphyses and flushing the medullary cavities with 1.Oml of Hank's solution (GIBCO, Grand Island, NY) injected into the medullary cavity through a no. 21 needle under pressure. Bone homogenate was prepared by removing 1 femur from donor mice, clipping off the epiphyseal ends, flushing the medullary cavity free of all marrow, splitting the diaphysis into halves and gently grinding them in a sterile mortar and pestle with 1.0 ml of Hank's solution. The paste obtained from one-half of a femur was then spread on one side of a CEM . Purified matrix components were similarly applied and spread uniformly Over one surface of a CEM. Fifteen-milligram quantities of each species were placed on a single CEM. Combination of collagen I and PG (15 mg quantities of each) were also placed on CEM and implanted i.p. and S.C. Prepamtion of Purified Mat& Proteins and PG
Type I Collagen from Murine Bone Femurs were decalcified by repeated changes of 1.0 M EDTA solutions at 4°C over 5 days. Bone residue (now C a m , free) was resuspended in 0.5 M acetic acid containing 1.0 mglml pepsin and digested at 4°C for 24 h. Supernatant (containing collagen) was precipitated by the addition of NaCl to a 5% final concentration. Precipitate was redissolved in 0.5 M acetic acid and reprecipitated with NaCl as above. Precipitate was washed with phosphate-buffered saline (PBS). Murine Cartilage Collagen flype II) and Proteoglycan Prepamtion Mouse sternae were suspended in 4.0 M guanidine chloride (Sigma Chemical Co., St. Louis, MO) and 0.05 M Tris, pH 7.4, for 24 h. Solubilized material (containing proteoglycans) and insoluble residue (containing type II collagen) were processed separately.
Matrix Proteins and Hematopoiesis
427
Collagen Residue was acidified with acetic acid and digested with 1.0 mg/ml pepsin (Sigma) at 4°C overnight. The residue was then neutralized and NaCl added to 3.5 M. This resulted in the solubilization of type II collagen. The soluble material was then reprecipitated and washed in PBS. Proteoglycans The 4 M guanidine chloride extract was mixed with 2 times its volume of cold 100% ethanol. The precipitate was collected and redissolved in PBS. Any remaining precipitate was diswded. The supernatant was precipitated as purified protmglycan with 100%ethanol.
Laminin Semipurifiedlaminin in the form of Matngel (Collaborative Research, Bedford, MA) was used in these experiments. This material is 70% laminin, 15% collagen IV, 10% heparin sulfate and 5% proteoglycan and eutactine. Collagen IV (Bovine Lens Protein) Bovine lens capsule was solubilized in 0.5 M acetic acid at 4U'C. The solubilized material was precipitated with 0.5 N NaCl which was then dialyzed to neutral. The precipitated material was centrifuged and resuspended in neutral phosphate buffer.
Results None of the CEM enriched with purified noncellular matrix species implanted either i.p. or S.C. developed bone or hematopoiesis. One unexpected observation was that CEM with collagen I and proteoglycan developed extensive sinusoid-like structures, but there was no hematopoiesis associated with these vessels (Fig. 1). Combinations of collagen I and proteoglycan did not enhance the development of sinusoids or induce bone or hematopoiesis. The CEM coated with purified matrix species became increasingly fibrotic with extensive collagen deposition. The negative control uncoated CEM implanted i.p. and S.C. were also devoid of any bone formation or hematopoiesis. These CEM became increasingly fibrotic compared with CEM with non-cellular matrix proteins and PGs. The positive controls coated with living stromal cells from bone or bone marrow developed extensive trilineal hematopoiesis and new bone formation after 6 and 12 months i.p. implantation [l].
Discussion These experiments were negative in the sense that they did not provide evidence for an inductive or modulatory role for hematopoiesis or bone formation for any of the matrix species studied. However, they do not exclude a role for other matrix factors such as the bone-inducing glycoprotein osteogenin present in bone and tooth matrix powder [6-81. We have demonstrated that stromal cells from bone or bone marrow secrete
Knospe/Husseini/Schwartz
428
Fig. 1. CEM coated with proteoglycanand implanted for 6 months i.p. Hematoxylin . is extensive sinusoid and eosin stain. Original magnification(A) 4x and (B) 4 0 ~There formation throughout the interior of the CEM (arrows). Hematopoiesisand bone are absent.
Matrix Proteins and Hematopoiesis
429
a similar factor which is adsorbed onto the enriched CEM after intervals of 1-6 months residence in the peritoneal cavity. Subcutaneously implanted CEM containing only non-cellular matrix factors rapidly induced new bone and hematopoiesis, but similar i.p. implanted CEM did not develop new bone or hematopoiesis [9, 101. Recently, we studied GAG and PG species in S.C. implanted tooth matrix and bone matrix. Initially, the implants contained abundant GAGs which appeared to be involved in the attraction of protomesenchymal cells resident only in skin and not in other tissues, including peritoneal cavity. These cells were transformed by osteogenin to become bone-forming cells and hematopoietic stromal cells. Later, the transformed bone cells formed proteoglycan species. The inactive bone matrix implants did not contain significant amounts of GAGs or PGs [lo-121. It is probable that PGs may modulate hematopoiesis, and yet not affect stromal cells [3-51. The observation that collagen I and proteoglycan seem to stimulate capillary or sinusoid-like development is interesting. Vascular cells alone may be ineffective in forming a hematopoietic microenvironment, but in concert with other stromal cells and noncellular matrix may be able to form a hematopoietic microenvironment [13, 141.
Acknowledgment This work was supported by a contract from the U.S. Office of Naval Research (contract number N00014-84-(2-0302).
References Knospe WH, Husseini SG, Adler SS. Hematopoiesis on cellulose ester membranes (CEM). In. Long-term histologic changes between 6 and 12 months after i.p. implantation. Exp Hematol 1983;ll:Sl2-521. McCuskey RS, Meineke HA, Pinkstaff CA,Knospe WH. Hematopoiesis on cellulose ester membranes (CEM). VI. Histochemical evaluation of stromal-CEM interactions after stromal enrichment. Exp Hematol 1984;12:25-30. Zuckerman KS,Wicha MS. Extracellular matrix production by the adherent cells of long-term munne bone marrow cultures. Blood 1983;61:540-547. Spooncer E, Gallagher JT, Krizsa F, Dexter TM. Regulation of haemopoiesis in longterm bone mamw cultures. IV.Glycosaminoglycan synthesis and the stimulation of haemopoiesis by 0-D-xylosides. J Cell Biol 1983;96:510-514. McCuskey PA, McCuskey RS,Meineke HA. Studies of the hemopoietic microenvironment. IV. In v i m microscopic and histochemical study of allograftsof bone marrow in the hamster cheek pouch. Exp Hematol 1975;3:297-308. Reddi AH, Huggins CB. Formation of bone marrow in fibroblast-transformation ossicles. Proc Natl Acad Sci USA 1975;72:2212-2216. Reddi AH. Collagen and cell differentation. In: Ramachandran GN, Reddi AH, eds. Biochemistry of Collagen. New York: Plenum Publishing Corp., 1976:449-478. Sampath TK,Muthukumaran N, Reddi AH. Isolation of osteogenin, an extracellular matrix-associated, bone inductiveprotein, by heparin affinity chromatography. Proc Natl Acad Sci USA 1987;84:7109-7ll3.
KnospeIHusseinilSchwartz
430
9 Knospe WH, Husseini SG. Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM. Exp Hematol 1986;14975-980. 10 Knospe WH, Husseini SG, Fried W. Hematopoiesis on cellulose ester membranes. XI. Induction of new bone and a hematopoietic microenvironment by matrix factors secreted by marrow stromal cells. Blood 1989;74:66-70. 11 Knospe WH, Husseini SG, Adler SS, Reddi AH. Decalcified tooth matrix powder induces new bone formation and hematopoietic microenvironment in the mouse. Int J Cell Cloning 1985;3:320-329. 12 McCuskey RS, Reddi AH, Knospe WH. Histochemical evaluation of the hemopoietic microenvironment induced by subcutaneous implants of powdered tooth matrix or bone in mice. Blood 1985;66(suppl 1):l32a. 13 Knospe WH, Blom J, Crosby WH. Regeneration of locally irradiated bone marrow. I. Dose dependent, long-term changes in the rat, with particular emphasis upon vascular and stromal reaction. Blood 1966;28:398-415. 14 Knospe WH, Blom J, Crosby WH. Aplastic anemia. Dependence of function on structure in the bone marrow. Blood 1967;30:851.
