Back to the future: moving beyond "mesenchymal stem cells"

The last decade was dominated by dissemination of the notion that postnatal "mesenchymal stem cells," found primarily in bone marrow but also in other tissues, can generate multiple skeletal and nonskeletal tissues, and thus can be exploited to regenerate a broad range of tissues and organs. The concept of "mesenchymal stem cells" and its applicative implications represent a significant departure from the solidly proven notion that skeletal stem cells are found in the bone marrow (and not in other tissues). Recent data that sharpen our understanding of the identity, nature, origin, and in vivo function of the archetypal "mesenchymal stem cells" (bone marrow skeletal stem cells) point to their microvascular location, mural cell identity, and function as organizers and regulators of the hematopoietic microenvironment/niche. These advances bring back the original concept from which the notion of "mesenchymal stem cells" evolved, and clarify a great deal of experimental data that accumulated in the past decade. As a novel paradigm emerges that accounts for many facets of the biology of skeletal stem cells, a novel paradigm independently emerges for their applicative/translational use. The two paradigms meet each other back in the future.     

"Ischemia/reperfusion" for stem cells of two "critical" cell renewal systems of organism

In this article is presented the result of the experiments on mice-hybrids F1(CBA x C57B1/6), which indicates the presence of the reaction of "ischemia/reperfusion" for stem cells of two "critical" cell renewal systems of organism (bone marrow and intestinal epithelium) during the irradiation under the conditions of hypoxic radioprotector application. The additional injection of the source of nitric oxide radicals-sodum nitroprusside (SNT) to the mice right after the irradiation under the conditions of hypoxic protection by serotonin, resulted the substantial increase of the survival rate of hematopoietic stem cells (registered by the methods of endogenous and exogenous colony forming in spleen) and stem cells of intestinal epithelium (registered by the method of intestinal "microcolonies"). The similar radioprotective effect was also registered during the test of survival rate of mice under tests of "bone marrow" and "intestinal" forms of radiation lethality that is evidence of the importance of the realization of phenomenon "ischemia/reperfusion" in the reaction of whole organism on the acute radiation injury. As SNP weakens the manifestation apoptosis and necrosis through competition with active forms of oxygen (AFO) during the period of "reperfusion" on basis of the found out phenomenon experimental model for studying mechanisms of stem cells damage in vivo induced by AFO and for the search of the modifiers weakening or strengthening such damage can be developed.     

"Matrigenin" activity from bovine bone--I. Partial purification of activity

1. Bovine bone contains an extractable activity which stimulated the synthesis of glycosaminoglycans by bovine synovial, human synovial and mouse 3T3 fibroblastic cells in culture. Human cells were used to develop an assay for purification of the stimulatory activity ("matrigenin" activity) from bovine bone. 2. Partial purification of "matrigenin" activity was achieved by precipitation of the EDTA extract at pH 3.5 and Sepharose CL-6B chromatography in 4 M guanidinium HCl. Dissociative conditions were necessary to prevent aggregation. 3. On SDS-polyacrylamide gel electrophoresis the activity ran with a mobility equivalent to a Mr = 27,500 and could be recovered from the SDS gels.     

Ultrastructural features of the plasma cells in "non-secretory" myeloma.

A case of "non-secretory" multiple myeloma is described. The diagnosis was based on the clinical picture, typical radiological findings, and infiltration of the bone marrow by myeloma cells which showed specific immunofluorescence staining mainly with antisera for IgM and kappa light chains. An attempt is made to explain the absence of pathological proteins in the serum, based on the ultrastructural findings of the myeloma cells, which showed "buddings" of the cell membranes containing endoplasmic reticulum and cytoplasmic material. It is suggested that the cells of the "non-secretory" type of multiple myeloma possess a normal excretory mechanism, but the pathological proteins are prevented to be secreted in the serum being surrounded by portions of the cell membrane.     

Frequency analysis of the antibody specificity repertoire of mitogen-reactive B cells and "spontaneously" occurring "background" plaque-forming cells in nude mice

The antibody specificity repertoire of lipopolysaccharide (LPS)-reactive B cells has been determined in the spleens and bone marrow (BM) of C57BL/Ka athymic nude mice using a limiting dilution culture system that allows the growth and development of every LPS-reactive B cell into a clone of IgM-secreting cells. In addition, the numbers of "spontaneously" occurring ("background") IgM-, IgG-, and IgA-secreting cells as well as the "background" IgM antibody specificity repertoire has been assessed in spleens and BM. The frequencies of antigen-specific LPS-reactive B cells of C57BL/Ka nude and thymus-bearing mice showed a great similarity and ranged from 1 in 1000 to 1 in 2500 for sheep red blood cells (SRBC), horse red blood cells (HRBC), and goat red blood cells (GRBC), from 1 in 10 to 1 in 25 for 5-iodo-3-nitrophenyl-coupled (SRBC), from 1 in 15 to 1 in 150 for 4-hydroxy-3,5-dinitrophenyl-coupled SRBC, and from 1 in 70 to 1 in 140 for 2,4,6-trinitrophenyl-coupled SRBC. The specificity repertoire of the "background" IgM-secreting cells differed from that of age-matched thymus-bearing controls and was different in young and old C57BL/Ka nude mice. Within the limitations of having assessed only a minor fraction of the total B-cell antibody specificity repertoire and supposing that nude mice are largely devoid of functional T cells, the data presented suggest that the generation of the specificity repertoire of newly-formed B cells is hardly or not affected by T cells. On the other hand, T cells do affect the expression of the established repertoire, represented by "background" immunoglobulin-secreting cells.     

Population dynamics of "null" and Thy1lo lymphocytes in mouse bone marrow: genesis of cells with natural killer cell lineage characteristics.

The population dynamics of "null" small lymphocytes lacking B and T lineage markers in mouse bone marrow have been examined using a combination of immunolabeling and hydroxyurea (HU) deletion techniques. The binding of the B lineage-associated mAb, 14.8, and anti-Thy1.2 to bone marrow cells has been detected radioautographically. Null cells lacking 14.8 and Thy1.2 determinants (14.8- Thy1-) formed a substantial subset (12-14%) of bone marrow small lymphocytes, representing 0.5 x 10(6) cells per femur (2-3% of nucleated cells). HU treatment revealed an exceptionally rapid turnover of the null small lymphocyte population (T1/2, 7.5 hr) compared with 14.8+ cells (T1/2, 20.5 hr) and Thy1+ cells (T1/2, 53 hr). Small lymphocytes bearing low intensities of Thy1 (Thy1lo) were also rapidly renewed (T1/2, 28 hr) whereas those with high intensities of Thy1 (Thy1hi) were renewed only slowly (T1/2, 123 hr). During ontogeny, null small lymphocytes first appeared in the fetal liver by Day 11 and the fetal spleen by Day 16, but increased rapidly in the bone marrow in early postnatal life. Double immunolabeling techniques demonstrated that 10% of null small lymphocytes in the bone marrow expressed NK1.1 antigen, while larger proportions bound to tumor (YAC.1) cells in vitro and displayed Fc receptors. The NK1.1-bearing fraction of null small lymphocytes in bone marrow was depleted by HU treatment only after an initial delay. NK1.1 was also expressed on subsets of Thy1lo cells and Thy1hi cells. The results have revealed the continuous production in mouse bone marrow of null and Thy1lo small lymphocytes, totaling 1-3 x 10(7) cells/day and 1.2 x 10(6) cells/day, respectively. The findings suggest that the large-scale production of null lymphocytes in mouse bone marrow includes the genesis of NK lineage cells which express NK1.1 and Thy1lo during a period of terminal maturation.     

Increased myeloid cell responses to M-CSF and RANKL cause bone loss and inflammation in SH3BP2 "cherubism" mice

While studies of the adaptor SH3BP2 have implicated a role in receptor-mediated signaling in mast cells and lymphocytes, they have failed to identify its function or explain why SH3BP2 missense mutations cause bone loss and inflammation in patients with cherubism. We demonstrate that Sh3bp2 "cherubism" mice exhibit trabecular bone loss, TNF-alpha-dependent systemic inflammation, and cortical bone erosion. The mutant phenotype is lymphocyte independent and can be transferred to mice carrying wild-type Sh3bp2 alleles through mutant fetal liver cells. Mutant myeloid cells show increased responses to M-CSF and RANKL stimulation, and, through mechanisms of increased ERK 1/2 and SYK phosphorylation/activation, they form macrophages that express high levels of TNF-alpha and osteoclasts that are unusually large. M-CSF and RANKL stimulation of myeloid cells that overexpress wild-type SH3BP2 results in similar large osteoclasts. This indicates that the mutant phenotype reflects gain of SH3BP2 function and suggests that SH3BP2 is a critical regulator of myeloid cell responses to M-CSF and RANKL stimulation.     

Detection of residual aneuploid leukemic cells by "continuous gating".

BACKGROUND: A new method for the detection of residual aneuploid leukemic cells in bone marrow by flow cytometry is described. This method is based on the analysis of FCM derived list-mode-datasets with a new software called "Continuous Gating". The program is able to decrease the detection level of aneuploid tumor cells by analyzing groups of cells with comparable antigen density and scatter properties. METHODS: Aneuploid acute lymphocytic leukemia cells with a known CD34 expression were diluted with diploid bone marrow cells to a concentration of 10, 1, 0.1, 0.05, and 0.01%. Each sample was measured in a FACScan flow cytometer, after staining with CD34 Moab and propidium iodide. Listmode-data were analyzed with the new "Windows"-based "Continuous Gating" software. A gate was set in the DNA parameter, defining the channels in which the aneuploid G0/G1-peak of possible residual tumor-cells should be found. Ten thousand overlapping gates of size 200 x 200 channels (out of 1,023 x 1,023 channels) were set automatically by the program into the side-scatter (SSC)/CD34 dot-plot, calculating the percentage of aneuploid G0/G1-phase cells for every specific gate. RESULTS: The results are plotted in a contour-plot. In dot-plot gates with less than 20 cells, the calculation of the percentage of aneuploid cells was declared invalid and the area in the contour-plot was marked. Detection of residual aneuploid cells, based on a defined expression of CD34 and granularity (SSC), was possible down to a contamination of 0.1%. CONCLUSIONS: The new "Continuous Gating" software can be used for the automated detection of aneuploid leukemic cells, if the density of a certain surface-marker is slightly different from normal cells.     

The skeletal attachment of tendons--tendon "entheses"

Tendon entheses can be classed as fibrous or fibrocartilaginous according to the tissue present at the skeletal attachment site. The former can be "bony" or "periosteal", depending on whether the tendon is directly attached to bone or indirectly to it via the periosteum. At fibrocartilaginous entheses, the uncalcified fibrocartilage dissipates collagen fibre bending and tendon narrowing away from the tidemark; calcified fibrocartilage anchors the tendon to the bone and creates a diffusion barrier between the two. Where there are additional fibrocartilaginous specialisations in the tendon and/or bone next to the enthesis, an "enthesis organ" is created that reduces wear and tear. Little attention has been paid to bone at entheses, despite the obvious bearing this has on the mechanical properties of the interface and the clinical importance of avulsion fractures. Disorders at entheses (enthesopathies) are common and occur in conditions such as diffuse idiopathic skeletal hyperostosis and the seronegative spondyloarthropathies. They are also commonly seen as sporting injuries such as tennis elbow and jumper's knee.     

From mechanostat theory to development of the "Functional Muscle-Bone-Unit"

Bone densitometric data are often difficult to interpret in children and adolescents because of large inter- and intraindividual variations in bone size. Here, we propose a functional approach to bone densitometry that addresses two questions: is bone strength normally adapted to the largest physiological loads, that is, muscle force? Is muscle force adequate for body size? The theoretical background for this approach is provided by the mechanostat theory, which proposes that bones adapt their strength to keep the strain caused by physiological loads close to a set point. Because the largest physiological loads are caused by muscle contractions, there should be a close relationship between bone strength and muscle force or size. The proposed two-step diagnostic algorithm requires a measure of muscle force or size and a measure of bone mineral content (BMC) at a corresponding location. The results can be combined into four diagnostic groups. In the first situation, muscle force or size is adequate for height. If the skeleton is adapted normally to the muscle system, the result is interpreted as "normal". If it is lower than expected for muscle force or size, a "primary bone defect" is diagnosed. In the second situation, muscle force or size is too low for height. Even if the skeleton is adapted adequately to the decreased mechanical challenge, this means that bone mass and presumably strength are still too low for body height. Therefore, a "secondary bone defect" is diagnosed. It is hoped that the more detailed insights thus gained could help to devise targeted strategies for the prevention and treatment of pediatric bone diseases.     

"Matrigenin" activity from bovine bone--III. Effects on glycosaminoglycans and proteoglycans of human synovial cells in culture

1. Human synovial fibroblastic cells were cultured in the presence and absence of an extract from bovine bone containing "matrigenin" activity. The rate of incorporation of radioactivity into the glycosaminoglycans of the medium of "matrigenin"-treated cultures increased after 24 hr of incubation, compared to "controls". 2. Higher serum concentrations had a greater effect on the incorporation of radioactivity into hyaluronic acid synthesized by "matrigenin"-treated cultures, than by "controls". 3. Incorporation of radioactive precursors into the proteoglycans isolated from the medium was greater in the "matrigenin"-treated cultures than in "controls". The synthesis of a large mol. wt proteoglycan was specifically stimulated.     

14 cases of "anti-N" antibodies in hemodialysis patients in Marseille

Some medical centers re-use dialysis units sterilized with formaldehyde. In a study of 239 cases, 14 "anti-N" antibodies were found only among the 59 patients of the medical centers which re-use dialysis units. The action of formol seems to be confirmed by the presence of "anti-N" in 2 patients who had undergone prosthesis several times, but not dialysis. For these prostheses, a bone cement, sterilized with formol, was used. These "anti-N" are very often associated with cold autoagglutinins, and appear regardless of the patient's MN group. The action of formaldehyde suggests the following hypotheses:--antigenic modification;--disturbances in the immune response mechanisms;--a combination of the two. In the first hypothesis: the action of formol discovered since a long time on red cells. In the second hypothesis: the existence of auto-agglutinins only among the 14 hemo-dialysis patients with anti-N antibodies.     

Heavy chain loss after treatment with Melphalan in a patient with "nonsecretory" myeloma

A case of "nonsecretory" myeloma is described. The patient had typical osteolytic lesions and marked infiltration of myeloma cells in the bone marrow, and plasma cell leukemia. A good partial remission was obtained with Melphalan, but the patient relapsed and died one year later. Immunofluorescent and immunoelectroscopic studies on the myeloma cells demonstrated the presence of cytoplasmic gamma-and kappa-chains at the initial stage and of only kappa-chains at a relapse. The electron microscopic method for polysome analysis indicated that both L-and H-chains were synthesized on membrane-bound polysomes initially, but the ability to produce H-chain was missing at the relapse.     

The effect of various stage maturity of erythrocytes upon proliferatio rate of erythroid cells in "crown" of erythroblastic islands

Joint cultivation of small amounts of young and mature erythrocytes with bone marrow erythroblastic islands during 24 hours in liquid culture system results in erythropoiesis stimulation due to increase of number of cells in the state of mitosis in island's "crown". Prolonged joint cultivation and/or joint cultivation with plenty of premature and mature erythrocytes leads to inhibition of the erythropoiesis in the state of mitosis in the island's "crown". The maximum of the inhibitory effect was shown by erythrocytes from polycythemia rats.     

"Culture shock" from the bone cell's perspective: emulating physiological conditions for mechanobiological investigations

Bone physiology can be examined on multiple length scales. Results of cell-level studies, typically carried out in vitro, are often extrapolated to attempt to understand tissue and organ physiology. Results of organ- or organism-level studies are often analyzed to deduce the state(s) of the cells within the larger system(s). Although phenomena on all of these scales—cell, tissue, organ, system, organism—are interlinked and contribute to the overall health and function of bone tissue, it is difficult to relate research among these scales. For example, groups of cells in an exogenous, in vitro environment that is well defined by the researcher would not be expected to function similarly to those in a dynamic, endogenous environment, dictated by systemic as well as organismal physiology. This review of the literature on bone cell culture describes potential causes and components of cell "culture shock," i.e., behavioral variations associated with the transition from in vivo to in vitro environment, focusing on investigations of mechanotransduction and experimental approaches to mimic aspects of bone tissue on a macroscopic scale. The state of the art is reviewed, and new paradigms are suggested to begin bridging the gap between two-dimensional cell cultures in petri dishes and the three-dimensional environment of living bone tissue. osteoblast; osteocyte; tissue engineering; mechanobiology; mechanochemical transduction; fluid flow     

Ultrastructural morphometric study of efferent nerve terminals on murine bone marrow stromal cells, and the recognition of a novel anatomical unit: the "neuro-reticular complex"

In order to extend our understanding of the role of nerve fibers in the structure and function of bone marrow stroma, we have examined nerve terminals, arterioles, and capillaries in femoral bone marrow tissues of 50 C57BL strain mice, using electron microscopy and morphometric methods. Within the adventitia of arterioles, a particular type of cell, termed periarterial adventitial (PAA) cell, is characterized by a thin veil-like cytoplasm which concentrically surrounds both nerves and arterioles. Nerve fibers containing both unmyelinated and myelinated axons are distributed mainly between the layers of PAA cells, but are found rarely on the sinus walls or within the hematopoietic parenchyma. Quantitatively, the efferent nerve terminals with many synaptic vesicles are distributed mainly beside arterial smooth muscle cells (Type I: 58.8%) or between the layers of PAA cells (Type III: 33.2%), and rarely in hematopoietic parenchyma (Type II: 5.3%) or on sinus walls (Type IV: 2.7%). In the case of Type II-IV nerve terminals, efferent (autonomic) nerves and bone marrow stromal cells which are connected by gap junctions (sinus adventitial reticular cells, intersinusoidal reticular cells, and PAA cells) appear to constitute a potential functional unit for signal conduction. We would like to propose a new term for this anatomical unit in marrow, the "neuro-reticular complex."     

Predominance of NK1.1+TCR alpha beta+ or DX5+TCR alpha beta+ T cells in mice conditioned with fractionated lymphoid irradiation protects against graft-versus-host disease: "natural suppressor" cells

We developed a nonmyeloablative host conditioning regimen in a mouse model of MHC-mismatched bone marrow transplantation that not only reduces radiation toxicity, but also protects against graft-vs-host disease. The regimen of fractionated irradiation directed to the lymphoid tissues and depletive anti-T cell Abs results in a marked change in the residual host T cells, such that NK1.1+ or DX5+asialo-GM1+ T cells become the predominant T cell subset in the lymphoid tissues of C57BL/6 and BALB/c mice, respectively. The latter "natural suppressor" T cells protect hosts from graft-vs-host disease after the infusion of allogeneic bone marrow and peripheral blood cells that ordinarily kill hosts conditioned with sublethal or lethal total body irradiation. Protected hosts become stable mixed chimeras, but fail to show the early expansion and infiltration of donor T cells in the gut, liver, and blood associated with host tissue injury. Cytokine secretion and adoptive transfer studies using wild-type and IL-4(-/-) mice showed that protection afforded by NK1.1+ and DX5+asialo-GM1+ T cells derived from either donors or hosts conditioned with lymphoid irradiation is dependent on their secretion of high levels of IL-4.     

"Delayed death" phenomenon: a synergistic action of cyclophosphamide and exogenous DNA

Morbidity and mortality in mice were observed upon administration of exogenous DNA following their pre-treatment with a cytostatic agent cyclophosphamide. Upon intraperitoneal injections, the fragments of exogenous DNA reached bone marrow cells. These cells were also found to internalize up to 1800 kb of exogenous DNA ex vivo. The 18-24 h time frame represents a final stage in the repair of DNA double-strand breaks, so when exogenous DNA was administered within this critical period of time, pathological changes were observed in many target organs. Namely, bone marrow cells underwent a sustained increase in apoptosis. Copy number of B1 and B2 DNA repeats in bone marrow cells remained unchanged, whereas in the control group of animals their levels were significantly decreased. Finally, the bone marrow cells of moribund animals completely lacked lymphoid progenitors, yet the CD34+ hematopoietic stem cell counts were normal. Histopathology analysis suggested that mice died due to accidental involution of lymphoid organs combined with a systemic inflammatory process induced by massive administration of exogenous DNA and depletion of lymphoid lineage.