Future cell-based therapies such as tissue engineering will benefit from a source of autologous pluripotent stem cells. For mesodermal tissue engineering, one such source of cells is the bone marrow stroma. The bone marrow compartment contains several cell populations, including mesenchymal stem cells (MSCs) that are capable of differentiating into adipogenic, osteogenic, chondrogenic, and myogenic cells. However, autologous bone marrow procurement has potential limitations. An alternate source of autologous adult stem cells that is obtainable in large quantities, under local anesthesia, with minimal discomfort would be advantageous. In this study, we determined if a population of stem cells could be isolated from human adipose tissue. Human adipose tissue, obtained by suction-assisted lipectomy (i.e., liposuction), was processed to obtain a fibroblast-like population of cells or a processed lipoaspirate (PLA). These PLA cells can be maintained in vitro for extended periods with stable population doubling and low levels of senescence. Immunofluorescence and flow cytometry show that the majority of PLA cells are of mesodermal or mesenchymal origin with low levels of contaminating pericytes, endothelial cells, and smooth muscle cells. Finally, PLA cells differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, the data support the hypothesis that a human lipoaspirate contains multipotent cells and may represent an alternative stem cell source to bone marrow-derived MSCs.

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Multilineage cells from human adipose tissue: implications for cell-based therapies.

Endothelial cells were isolated from freshly obtained human umbilical cords by collagenase digestion of the interior of the umbilical vein. The cells were grown in tissue culture as a homogeneous population for periods up to 5 mo and some lines were subcultured for 10 serial passages. During the logarithmic phase of cell growth, cell-doubling time was 92 h. Light, phase contrast, and scanning electron microscopy demonstrated that cultured human endothelial cells grew as monolayers of closely opposed, polygonal large cells whereas both cultured human fibroblasts and human smooth muscle cells grew as overlapping layers of parallel arrays of slender, spindle-shaped cells. By transmission electron microscopy, cultured endothelial cells were seen to contain cytoplasmic inclusions (Weibel-Palade bodies) characteristic of in situ endothelial cells. These inclusions were also found in endothelial cells lining umbilical veins but were not seen in smooth muscle cells or fibroblasts in culture or in situ. Cultured endothelial cells contained abundant quantities of smooth muscle actomyosin. Cultured endothelial cells also contained ABH antigens appropriate to the tissue donor's blood type; these antigens were not detectable on cultured smooth muscle cells or fibroblasts. These studies demonstrate that it is possible to culture morphologically and immunologically identifiable human endothelial cells for periods up to 5 mo.

/pdf/culture-of-human-endothelial-cells-derived-from-umbilical-3vt4ihjkgj.pdf

Culture of Human Endothelial Cells Derived from Umbilical Veins. IDENTIFICATION BY MORPHOLOGIC AND IMMUNOLOGIC CRITERIA

The pathogenesis of atherosclerosis (first of two parts).

A permanent human cell line, EA . hy 926, has been established that expresses at least one highly differentiated function of vascular endothelium, factor VIII-related antigen. This line was derived by fusing human umbilical vein endothelial cells with the permanent human cell line A549. Hybrid cells that survived in selective medium had more chromosomes than either progenitor cell type and included a marker chromosome from the A549 line. Factor VIII-related antigen can be identified intracellularly in the hybrids by immunofluorescence and accumulates in the culture fluid. Expression of factor VIII-related antigen by these hybrid cells has been maintained for more than 100 cumulative population doublings, including more than 50 passages and three cloning steps. This is evidence that EA . hy 926 represents a permanent line.

https://www.pnas.org/content/pnas/80/12/3734.full.pdf

Permanent cell line expressing human factor VIII-related antigen established by hybridization

Endothelial cells, which form the inner cellular lining of blood vessels and lymphatics, display remarkable heterogeneity in structure and function. This is the first of a 2-part review focused on phenotypic heterogeneity of blood vessel endothelium. This review provides an historical perspective of our understanding of endothelial heterogeneity, discusses the scope of phenotypic diversity across the vascular tree, and addresses proximate and evolutionary mechanisms of endothelial cell heterogeneity. The overall goal is to underscore the importance of phenotypic heterogeneity as a core property of the endothelium.

Phenotypic Heterogeneity of the Endothelium: I. Structure, Function, and Mechanisms

Antihemophilic factor (AHF, Factor VIII) antigen has been demonstrated in cultured human endothelial cells by immunofluorescence studies using monospecific rabbit antibody to human AHF. Control studies with cultured human smooth muscle cells and human fibroblasts were negative. By radioimmunoassay it was demonstrated that cultured human endothelial cells contain AHF antigen which is released into the culture medium. Cultured smooth muscle cells and fibroblasts did not have this property. Cultured endothelial cells incorporated radioactive amino acids into high molecular weight, AHF antigen-rich protein fractions prepared from the culture media, 7% of the radioactive amino acid counts incorporated into this material were precipitated by globulin prepared from rabbit anti-AHF whereas normal rabbit globulin precipitated only 1.5% of the counts. Although cultured endothelial cells actively synthesize AHF antigen, AHF procoagulant activity was not detected in the culture medium. Studies seeking a basis for the lack of procoagulant activity have not clarified this deficiency, but they have established that exogenous AHF procoagulant activity is not inactivated by the tissue culture system.

/pdf/synthesis-of-antihemophilic-factor-antigen-by-cultured-human-42esij6igt.pdf

Synthesis of Antihemophilic Factor Antigen by Cultured Human Endothelial Cells

Cultured human endothelial cells synthesize and secrete a protein(s) which has Factor VIII antigen but which lacks Factor VIII clot-promoting activity (J. Clin. Invest. 52, 2757-2764, 1973). Von Willebrand factor activity has been identified in medium from cultured human endothelial cells. This activity was demonstrated by the ability to correct the defect in platelet adhesiveness of blood obtained from patients with von Willebrand's disease. This activity also supported ristocetin-induced aggregation of washed normal human platelets. The von Willebrand factor activity from cultured endothelial cells has physicochemical and immunologic properties like those of the von Willebrand factor activity and the Factor VIII antigen present in human plasma and the Factor VIII antigen synthesized by human endothelial cells in vitro. Rabbit antibody to chromatographic fractions containing endothelial cell von Willebrand factor inhibits the platelet retention of normal blood in glass bead columns.

https://www.pnas.org/content/pnas/71/5/1906.full.pdf

Synthesis of von Willebrand Factor by Cultured Human Endothelial Cells

The tissue localization of antihemophilic factor (AHF, Factor VIII) has been determined by immunofluorescent studies using monospecific rabbit antibody to human AHF. Specific staining demonstrating AHF antigens has been identified in endothelial cells of a wide range of human tissues. The staining pattern was observed in endothelial cells of arteries, capillaries, and veins as well as the cells lining hepatic and splenic sinusoids. Specific fluorescence was limited to these endothelial cells in sections of kidney, liver, spleen, lymph node, cardiac and smooth muscle, thyroid, umbilical cord, and skin. Absorption studies established that the staining was specific for cells in which there were proteins that had AHF antigens. The demonstration of fluorescence within the cytoplasm of endothelial cells suggests that these cells synthesize proteins that have AHF antigens.

Antihemophilic factor antigen. Localization in endothelial cells by immunofluorescent microscopy.

Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site.

Leon W. Hoyer

Papers

Synthesis of Antihemophilic Factor Antigen by Cultured Human Endothelial Cells

Synthesis of von Willebrand Factor by Cultured Human Endothelial Cells

Antihemophilic factor antigen. Localization in endothelial cells by immunofluorescent microscopy.

Some factor VIII inhibitor antibodies recognize a common epitope corresponding to C2 domain amino acids 2248 through 2312, which overlap a phospholipid-binding site.

Prevention and treatment of factor VIII inhibitors in murine hemophilia A.