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.

5'-Noncoding sequences have been compiled from 699 vertebrate mRNAs. (GCC) GCCA/GCCATGG emerges as the consensus sequence for initiation of translation in vertebrates. The most highly conserved position in that motif is the purine in position -3 (three nucleotides upstream from the ATG codon); 97% of vertebrate mRNAs have a purine, most often A, in that position. The periodical occurrence of G (in positions -3, -6, -9) is discussed. Upstream ATG codons occur in fewer than 10% of vertebrate mRNAs-at-large; a notable exception are oncogene transcripts, two-thirds of which have ATG codons preceding the start of the major open reading frame. The leader sequences of most vertebrate mRNAs fall in the size range of 20 to 100 nucleotides. The significance of shorter and longer 5'-noncoding sequences is discussed.

An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs

Publisher Summary This chapter describes two types of purification methods for Cathepsin B, Cathepsin H, and Cathepsin L. Method I is applicable to large amounts of frozen tissues, whereas method II is used with flesh tissue and takes advantage of a 50-fold purification factor attainable by isolation of lysosomes: it has the further advantage of separating the enzymes from inhibitors that are present in the cytosol and plasma. In first purification method, cathepsins B and H are purified from human liver. Method II involves purification of Cathepsins B, H, and L from rat liver. Method I include: extraction, autolysis, and acetone fractionation and DEAE-cellulose chromatography. The pool of cathepsin B from DEAE-cellulose is further purified by covalent chromatography on a column of aminophenylmercuric acetate coupled to Sepharose. Method II include: homogenization and cell fractionation gel; chromatography on Sephadex G-75; CM-Sephadex chromatography; chromatography of cathepsin L on concanavalin A-Sepharose. Cathepsin B can be with BZ-DL-Arg-NPhNO2 or Bz-Arg-2-NNap as substrate, wheras, Cathepsin H can be assayed selectively by use of an unblocked substrate such as Leu-NNap, Arg-NNap, or Arg-NMec. Three synthetic substrates have been used for cathepsin L assay: Bz-Arg-NH2, Z-Lys-OPhNO2, and Z-Phe-Arg-NMec.

Cathepsin B, Cathepsin H, and cathepsin L.

Osteoporosis is widely viewed as a major public health concern, but the exact magnitude of the problem is uncertain and likely to depend on how the condition is defined. Noninvasive bone mineral measurements can be used to define a state of heightened fracture risk (osteopenia), or the ultimate clinical manifestation of fracture can be assessed (established osteoporosis). If bone mineral measurements more than 2 standard deviations below the mean of young normal women represent osteopenia, then 45% of white women aged 50 years and over have the condition at one or more sites in the hip, spine, or forearm on the basis of population-based data from Rochester, Minnesota. A smaller proportion is affected at each specific skeletal site: 32% have bone mineral values this low in the lumbar spine, 29% in either of two regions in the proximal femur, and 26% in the midradius. Although this overall estimate is substantial, some other serious chronic diseases are almost as common. More importantly, low bone mass is associated with adverse health outcomes, especially fractures. The lifetime risk of any fracture of the hip, spine, or distal forearm is almost 40% in white women and 13% in white men from age 50 years onward. If the enormous costs associated with these fractures are to be reduced, increased attention must be given to the design and implementation of control programs directed at this major health problem.

/pdf/perspective-how-many-women-have-osteoporosis-59dbcqkyvh.pdf

Perspective : how many women have osteoporosis ?

Cells from fetal or neonatal skeleton can synthesize bone-like tissue in vitro In contrast, formation of bone-like tissue in vitro by cells derived from adult animals has rarely been reported and has not been achieved using cells from bone marrow We have explored development of bone-like tissue in vitro by bone marrow stromal cells Marrow stromal cells obtained from 40-43-day-old Wistar rats were grown in primary culture for 7 days and then subcultured for 20-30 days Cells were cultured in either alpha-minimal essential medium containing 15% fetal bovine serum, antibiotics, and 50 micrograms/ml ascorbic acid, or the above medium supplemented with either 10 mM Na-beta-glycerophosphate, 10(-8) M dexamethasone, or a combination of both Cultures were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry (for alkaline phosphatase activity), immunohistochemistry (for collagen type, osteonectin, and bone Gla-protein), scanning and transmission electron microscopy, energy dispersive X-ray microanalysis, and X-ray diffraction Collagenous, mineralized nodules exhibiting morphological and ultrastructural characteristics similar to bone were formed in the cultures, but only in the presence of both beta-glycerophosphate and dexamethasone Cells associated with the nodules exhibited alkaline phosphatase activity The matrix of the nodules was composed predominantly of type-I collagen and both osteonectin and Gla-protein were present X-ray microanalysis showed the presence of Ca and P, and X-ray diffraction indicated the mineral to be hydroxyapatite The nodules were also examined for bone morphogenetic protein-like activity Paired diffusion chambers containing partly demineralized nodules and fetal muscle were implanted intraperitonealy in rats Induction of cartilage in relation to muscle was observed histologically after 40 days in the chambers This finding provided further support for the bone-like nature of the nodules The observations show that bone-like tissue can be synthesized in vitro by cells cultured from young-adult bone marrow, provided that the medium contains both beta-glycerophosphate and, particularly, dexamethasone

Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats.

Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately two fold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activities were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activities was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in the PT cells. Parathormone stimulated acid phosphatase...

Biochemical Characterization with Parathormone and Calcitonin of Isolated Bone Cells: Provisional Identification of Osteoclasts and Osteoblasts

Six populations of bone cells (populations 1-6) were obtained by sequential digestion of mouse calvaria with collagenase and trypsin. After release from the tissue, each cell population was cultured for seven days. Parathormone, but not calcitonin, elicited an increase in intracellular cyclic AMP in the cells of populations 4, 5, and 6. In contrast, both hormones elicited increases in cyclic AMP in populations 2 and 3 but had no effect on population 1. When the cells of population 2 were exposed to a Falcontissue culture polystyrene surface for periods of time up to 5 min, many cells adhered. The nonadhering cell population contained a lesser proportion of cells responsive to calcitonin, whereas the adhering population contained a greater proportion responsive to this hormone. Conversely, when the cells of population 2 were exposed to an acid-treated nylon surface, the nonadhering cells contained a larger proportion of those responsive to calcitonin and a smaller proportion responsive to parathormone. When those cells that were enriched for calcitonin responsiveness were examined, we found an increased proportion that exhibited an asymmetric bipolar morphology. These differed from large amorphous, often binucleate, cells which predominated in those populations that responded exclusively to parathormone. These results establish that bone contains at least two types of target cells--one that responds to parathormone but not calcitonin, the other that responds predominantly to calcitonin.

https://www.pnas.org/content/pnas/72/8/3167.full.pdf

Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces

BONE growth and remodelling depend on resorption and deposition at the local level. The balance between these two processes seems to be modulated by the peptide hormones PTH and calcitonin. Parathyroid hormone (PTH) has been shown to induce bone resorption both in vivo1 and in vitro2, whereas calcitonin (CT) inhibits this process3. Recent studies suggest that both agents act on cellular mechanisms through a second messenger. Thus, it has been demonstrated that exposure of mouse calvaria to PTH or CT can lead to an increase in cyclic AMP content4,5. In addition, dibutyryl cyclic AMP can by itself induce bone resorption2.

Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells

Secretory protein-I (SP-I) of parathyroid glands and chromogranin A (CGA) of adrenal medullary chromaffin cells are chemically similar if not identical proteins. Both proteins are contained within secretory granules and appear to be cosecreted with granule contents, for example, in the parathyroid with PTH and in the adrenal with epinephrine and dopamine β-hydroxylase. Antisera to bovine SP-I and porcine CGA, together with antisera to a variety of peptide hormones, were used in an immunofluorescence study of rat tissues in order to determine the probable distribution and cellular localization of these proteins. In addition to their previously demonstrated presence in parathyroid and adrenal cells, the SP-I/CGA protein family was detected in cells of the thyroid that contained calcitonin and often SRIF but not thyroglobulin; in cells of the anterior pituitary staining for the α-subunit of TSH/FSH/LH but not in cells staining for GH, PRL, ACTH, or β-endorphin; in pancreatic islet cells staining for SRIF and...

Selective localization of the parathyroid secretory protein-I/adrenal medulla chromogranin A protein family in a wide variety of endocrine cells of the rat.

The actions of 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and parathormone, both effective bone-resorptive agents in vivo and in vitro, were tested on CT (osteoclast-like) and PT (osteoblast-like) bone cells maintained in culture. Both agents stimulated acid phosphatase activity and hyaluronate synthesis in the CT cells and decreased alkaline phosphatase, citrate decarboxylation, and collagen synthesis in the PT cells. Calcitonin inhibited the changes induced in the CT but not in the PT cells. The activity of 1,25-(OH)2D3 differed from that of parathormone in one key respect: it did not increase cellular cyclic adenosine monophosphate, whereas parathormone did. Prior incubation of the bone cells with 1,25-(OH)2D3 for 6 to 24 hours made the cells refractory to the effect of parathormone on cyclic adenosine monophosphate formation. These data suggest that 1,25-(OH)2D3 and parathormone induce bone resorption by affecting the same cell types (osteoblasts and osteoclasts) although at different cellular sites.

https://science.sciencemag.org/content/sci/197/4304/663.full.pdf

David V. Cohn

Papers

Biochemical Characterization with Parathormone and Calcitonin of Isolated Bone Cells: Provisional Identification of Osteoclasts and Osteoblasts

Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces

Separation of parathyroid hormone and calcitonin-sensitive cells from non-responsive bone cells

Selective localization of the parathyroid secretory protein-I/adrenal medulla chromogranin A protein family in a wide variety of endocrine cells of the rat.

1,25-dihydroxycholecalciferol and parathormone: effects on isolated osteoclast-like and osteoblast-like cells.