Immunologically mediated aplastic anemia in mice: evidence of hematopoietic stromal injury and injury to hematopoietic stem cells.

Immunologically mediated aplastic anemia (AA) results when lymph node cells (LNC) from C3H/He mice are injected intravenously (i.v.) into H-2 identical CBA/J mice previously given 600 cGy sublethal total-body gamma irradiation (TBI). Previously, we showed that T lymphocytes injure pluripotent hematopoietic stem cells and cause severe pancytopenia and death in 80 to 100% of mice within 3 to 4 weeks, with changes in the bone marrow suggesting stromal injury. The following models were used to study the stroma: (1) Transplantation of femurs from AA mice into normal syngeneic CBA/J mice. After 6 weeks, colony-forming unit-spleen (CFU-S) levels in the femur implants were measured in both AA and control mice (600 cGy TBI only). (2) Development of Dexter long-term bone marrow cultures from AA and control mice, which were used to support hematopoietic bone marrow cells (colony-forming units-granulocyte/macrophage [CFU-GM]) from normal mice. (3) Cellulose ester membranes (CEM) were coated with hematopoietic stroma from AA and control mice and then implanted intraperitoneally (i.p.) into syngeneic CBA/J mice. Six months later, the CEM were removed and analyzed for the presence of trilineal hematopoiesis and bone. Injury to the hematopoietic stroma was documented by the following: (1) Femurs from AA mice had a decreased number of CFU-S compared to controls; (2) Dexter cultures from AA mice formed abnormal stromal layers with a decreased capacity to support CFU-GM from normal donor mice; and (3) CEM coated with stromal cells from AA mice had a decreased capacity to support trilineal hematopoiesis and bone compared to CEM coated with marrow stroma from control mice.

Hematopoiesis on cellulose ester membranes. XIII. A combination of cloned stromal cells is needed to establish a hematopoietic microenvironment supportive of trilineal hematopoiesis.

A mixture of stromal cells from murine bone marrow placed upon cellulose ester membranes (CEM) and then implanted intraperitoneally (i.p.) in mice results in a regenerated hematopoietic microenvironment which supports trilineal hematopoiesis. We used this model to study the capacity of 5 cloned murine stromal cell lines of marrow origin to support hematopoiesis in vivo: MBA-1 (fibroblast); MBA-2 (endothelial); MBA-13 (fibroendothelial); 14F1.1 (endothelial-adipose); and 14M1.4 (macrophage).10(7) stromal cells of a single cell line were applied to 1.5 cm2 CEM, which were folded into tubes and implanted i.p. into mice. Similarly, combinations of 4, 3 and 2 stromal cell lines were applied to CEM and implanted i.p. Single lines were implanted into syngeneic hosts of the same murine strain from which the clone was derived and into nude mice. Combinations of stromal cells were implanted only in nude mice to avoid allogeneic incompatibility. CEM implants were removed after intervals of 5 to 36 weeks and examined histologically. 1) Stromal cells of a single phenotype did not develop hematopoiesis. 2) A combination of 4 stromal phenotypes (MBA-1, MBA-2, MBA-13 and 14F1.1) formed a hematopoietic microenvironment supportive of trilineal hematopoiesis and bone. 3) The combination of 14F1.1 (endothelial adipose) + a second stromal phenotype--MBA-1 (fibroblast) or MBA-2 (endothelial) or MBA-13 (fibroendothelial) also supported trilineal hematopoiesis and bone. 4) CEM coated with MBA-13 or MBA-1 developed bone but no hematopoiesis. The endothelial-adipose phenotype appears to be essential to support hematopoiesis but requires other types of stromal cells--fibroblast, fibroendothelial or endothelial phenotype.

Hematopoiesis on cellulose ester membranes (CEM). XII. Effect of membrane enrichment by purified matrix proteins.

Cellulose ester membranes (CEM) were enriched with the following purified 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-enriched CEM showed evidence of increased sinusoid-like vascular structures.

Hematopoiesis on cellulose ester membranes. XI. Induction of new bone and a hematopoietic microenvironment by matrix factors secreted by marrow stromal cells.

Cellulose ester membranes (CEM) were coated with stromal cells from bone marrow (BM) or bone and implanted intraperitoneally (IP) in CAF1 mice for intervals of 1 to 6 months. Previous studies indicated that matrix factors [glycoproteins (GPs), proteoglycans (PGs), and glycosaminoglycans (GAGs)] were secreted by the regenerating stromal cells and adsorbed by the CEM. After 1 to 6 months, the CEMs were removed, scraped free of adherent cells, and irradiated in vitro with 40 Gy. The scraped and irradiated CEMs were then reimplanted IP or subcutaneously (SC) for periods of 1 to 6 months in secondary syngeneic murine hosts. They were then removed for histologic study. CEMs reimplanted in SC sites developed bone and hematopoiesis as early as 1 month after implantation. Maximum hematopoiesis and bone formation was observed after 3 months. CEMs coated during the initial implantation with bone-derived stromal cells contained more bone and hematopoietic cells than did CEMs coated with marrow-derived stromal cells after SC implementation. Neither the CEMs coated with bone stromal cells nor those coated with marrow stromal cells developed new bone or trilineal hematopoiesis after being implanted IP. A few CEMs contained small foci of granulopoiesis only. We conclude that noncellular matrix substances deposited on CEMs by bone, and to a lesser degree by marrow cells, can induce prestromal cells in the SC tissues to produce a microenvironment suitable for trilineal hematopoiesis.

The ultrastructure of hematopoietic stroma on cellulose ester membranes implanted intraperitoneally into Sl/Sld and Sl+/Sl+ mice.

The structural features of hematopoietic stromal elements forming on cellulose ester membranes (CEM) implanted intraperitoneally into hematopoietically impaired, anemic Sl/Sld mice and their normal Sl+/Sl+ littermates were compared by combined light and electron microscopy. The generally thicker, multilayered stroma lining the Sl+/Sl+ CEM implants developed from a bed--a syncytium--of large, highly pleomorphic macrophage-type lining cells whose filopodial extensions exhibited extensive interactions (i.e., nurse cell interactions) with both stromal and hematopoietic elements. In contrast, the thinner stromal layers lining the CEM of Sl/Sld mice formed from a base of dysplastic lining elements. These CEM-lining macrophage-type cells had much reduced cytoplasmic volumes, less extensive interactive surface projections, and an absence of select types of cytoplasmic organelles (e.g., membrane-bound crystalline inclusions). These observations suggest that the reduction of cell layering and, in turn, hematopoietic support activity, is due to an impaired interactive capacity of these elemental lining cells, i.e., pleomorphic macrophagic cell types, in the hematopoietically impaired strain of Sl/Sld mice.

Hematopoiesis on cellulose ester membranes (CEM). IX. Enrichment of membranes with adherent layers from long-term marrow culture from normal or drug-treated mice (hematopoietic microenvironment study II).

Cellulose ester membranes (Millipore) or polytetrafluoroethylene (Mitex) membranes were coated with adherent layers taken from Dexter-type long-term cultures, 4-5, 8 or 12 weeks after initiation of culture. The cultures were established with marrow taken from untreated mice or, in some cases, from mice treated with a single lethal dose (LD10) of carmustine (BCNU) or cyclophosphamide. In the studies using untreated mice, the cultures went for 8 or 12 weeks and in the drug studies, for 4-5 weeks. The 8 and 12 week cultures were reseeded at 4 weeks. The membranes were implanted into the peritoneal cavities of mice for 3-12 months after which they were removed, fixed, sectioned and stained for histologic study. After 6 months of implantation, about 40% of the membranes coated with cells from non-drug-treated mice and 60% of the membranes coated with cells from drug-treated mice contained hematopoietic elements; often there were foci of trilineal hematopoiesis. Hematopoiesis never occurred without bone formation, but the reverse was not true. Membranes coated with adherent layers established from marrow of mice treated with cyclophosphamide or BCNU showed two main characteristics: 1) they supported hematopoiesis normally, and 2) the regeneration of stroma and hematopoiesis occurred earlier than in membranes coated with stroma derived from normal mice, perhaps because the cells from the drug-treated mice spent a shorter time in culture. In vitro culture may damage cells required to condition the membrane for hematopoiesis.

Hematopoiesis on cellulose ester membranes (CEM). X. Effects of in vitro irradiation of stromal cells prior to application on CEM.

Cellulose ester membranes (CEM) were coated with stromal cells from murine bone or bone marrow irradiated in vitro with 1000, 2000, or 4000 rad and then implanted i.p. in CAF1 mice for periods of six and 12 months. CEM coated with stromal cells from bone showed excellent regeneration of bone and hematopoiesis after 1000 rad in vitro irradiation. After 2000 rad, hematopoietic and bone regeneration was reduced by about 50%, and after 4000 rad it was completely absent in CEM coated with stromal cells from bone. CEM coated with stromal cells from bone marrow showed no regeneration of hematopoiesis or bone after 1000, 2000, and 4000 rad in vitro irradiation and residence i.p. for six and 12 months. These results indicate that regeneration of the hematopoietic microenvironment is dependent upon living stromal cells. A difference in radiation sensitivity is demonstrated between stromal cells from bone and from bone marrow.

Hematopoiesis on cellulose ester membranes: VII. Ultrastructure of stroma of marrow-enriched membranes with trilineal hematopoiesis.

The ultrastructure of developing osseous and hematopoietic tissue and supporting stroma was examined within intraperitoneally implanted, marrow-coated cellulose ester membranes (CEMs). During initial periods of implantation (two weeks), coated CEMs were shallowly infiltrated and surface-lined with mainly two stromal cell types--primitive mesenchymal cells and large, pleomorphic, multinucleated monocytoid-like stromal cells--and, in addition, endothelial cells in early stages of vessel development. Selective proliferation, maturation, and orientation of these cell types along the CEM's surface (one month), resulted in the formation of primitive osseous and hematopoietic tissue sites. Osseous sites developed as undifferentiated mesenchymal cells transformed into well-differentiated secretory cells residing in an electron-dense, pre-mineralized extracellular matrix, that upon extended implantation (3-6 months) formed bone. Hematopoietic sites developed as mesenchymal cells, in intimate contact with the monocytoid-like stromal cells, extended elongated branches into the medullary cavity, and enveloped newly formed surface-associated vascular structures. With subsequent maturation of vascular sinuses and supporting adventitial stroma (3-6 months), the sites were colonized with either uni-, bi-, or trilineal hematopoietic elements. These observations provide evidence that common, marrow-derived precursor stromal cells, i.e., mesenchymal and multinucleated monocytoid-like cells, cooperate in the development of both osseous and hemic tissue sites.

Decalcified tooth matrix powder induces new bone formation and hematopoietic microenvironment in the mouse.

Implants of bone and tooth matrix powder were placed subcutaneously (s.c.) on intraperitoneal (i.p.) Mitex or Polyvic membranes. Implants were removed for histology after 1-24 weeks. Macrophages, fibroblasts, and vascular sinusoids infiltrated around bone and tooth matrix particles after one week. In the s.c. tooth matrix implants, a few sites of cartilage formation and ossification developed at two weeks, and by three weeks granulocytopoiesis and megakaryocytopoiesis developed adjacent to new bone; erythropoiesis was not observed. In s.c. bone matrix implants and in the i.p. artificial membranes coated with bone or tooth matrix powder, ossification or hematopoiesis was not observed. Small numbers of CFU-s, CFU-nm, BFU-e, and CFU-e appeared 10-20 days after s.c. implantation of tooth matrix; none were detected in s.c. bone matrix implants.

Hematopoiesis on cellulose ester membranes (CEM). VIII. Studies of the hematopoietic microenvironment developing on intraperitoneally implanted CEM in S1/S1d and S1+/S1+ mice.

Cellulose ester membranes (CEM) were implanted intraperitoneally into S1/S1d and S1+/ S1+ mice. These CEM rapidly became coated with peritoneal cells capable of supporting primarily granulocytic colony development after seven days. S1/S1d-coated CEM showed a diminished capacity to support colony development compared with S1+/ S1+ CEM, perhaps reflecting the defect in the hematopoietic microenvironment of these mice. Marrow cells from S1+/S1+ and S1/S1d mice generated similar numbers of colonies on S1+/S1+ CEM. When CEM were transferred from a primary to a secondary host there was a tendency to remodel the CEM toward the characteristics of the secondary host. Peritoneal cells coating CEM from S1/S1d mice had less phagocytosis of yeast particles than peritoneal cells from S1+/S1+ mice. The cell coat on the membranes from S1/S1d mice was fewer cell layers in thickness than those on membranes coated in S1+/ S1+ mice.

Hematopoiesis on cellulose ester membranes (CEM). IV. Effect of variation in membrane composition and pore size.

Artificial membranes composed of mixed cellulose nitrate and acetate esters (MCEM), polytetrafluoroethylene (Mitex), cellulose acetate (Celotate), polyvinylchloride (Polyvic), polycarbonate (Nuclepore), or linear polyethylene with pore sizes ranging from 0.45 mu to 70 mu were coated on their inner surfaces with a paste of bone marrow, diaphyseal bone, or cells proliferating in vitro on the inner surfaces of curetted femurs obtained from syngeneic mice. Squares of these membranes were folded into thirds, stapled to form an open-ended, tube-like structure, and implanted i.p. into mice. At intervals of 6-12 months, the membranes were removed and studied histologically. Control membranes not coated with any substance were also studied. Trilineal hematopoiesis, bone formation, sinusoidal structures, fat cells, and hemosiderin-laden macrophages were found with all three coatings in the tubes formed from the MCEM or Mitex membranes. The Celotate and Polyvic membranes coated with bone cells and the Polyvic membranes coated with marrow also developed trilineal hematopoiesis and new bone tissue. The remaining membranes essentially had only fibrous tissue, except for rare foci of hematopoiesis which developed on an occasional 30 mu (but not 70 mu) pore size linear polyethylene membrane. The results of these studies suggest that hematopoiesis and bone cell formation on these artificial membranes is closely linked, and that there may be some interaction between membrane constituents and the cells destined to form the hematopoietic support layer in the fostering of hematopoiesis. Whether this interaction is stimulatory or inhibitory was not defined by these studies.

Hematopoiesis on cellulose ester membranes (CEM). V. Enrichment of CEM by demineralized mouse bone matrix powder.

Mouse bone matrix powder was implanted subcutaneously or put into folded tubular cellulose ester membranes (CEM) and implanted i.p. Calcification and new bone formation did not develop. Sinusoidal vascularization of the matrix developed by 3 to 4 wk. Stromal cells such as fat cells and fibroblasts were present by 4 wk. Hematopoiesis was absent except for rare foci of granulopoiesis on some of the CEM at 6 wk. Rat bone matrix implanted subcutaneously into mice and mouse bone matrix implanted subcutaneously into rats failed to induce new bone formation or hematopoiesis.

Hematopoiesis on cellulose ester membranes (CEM): III. Long-term histologic changes between 6 and 12 months after i.p. implantation.

Cellulose ester membranes (CEM) of pore size 0.45 mu were enriched on their inner surfaces with spleen or bone marrow or bone or regenerating primitive endosteal tissue folded into a tubular configuration and implanted i.p. Although CEM with enrichment with bone or marrow or spleen supported trilineal hematopoiesis 3 to 6 months after i.p. implantation, CEM at 8 and 9 months were progressively fibrotic and involuted with minimal hematopoietic support capacity. CEM of pore size 3 mu were enriched with bone marrow or bone or regenerating endosteal tissue. These CEM after 9 and 12 months supported excellent trilineal hematopoiesis and were always associated with osteogenesis. Regenerating primitive endosteal tissue is an equally effective source of stroma to regenerate the hematopoietic microenvironment. Unenriched CEM became progressively more fibrotic after 6 months.