Surface antigenic profiling of stem cells from human omentum fat in comparison with subcutaneous fat and bone marrow

Omentum fat derived stem cells have emerged as an alternative and accessible therapeutic tool in recent years in contrast to the existing persuasive sources of stem cells, bone marrow and subcutaneous adipose tissue. However, there has been a scanty citation on human omentum fat derived stem cells. Furthermore, identification of specific cell surface markers among aforesaid sources is still controversial. In lieu of this existing perplexity, the current research work aims at signifying omentum fat as a ground-breaking source of stem cells by surface antigenic profiling of stem cell population. In this study, we examined and compared the profiling of cell surface antigenic expressions of hematopoietic stem cells, mesenchymal stem cells, cell adhesion molecules and other unique markers such as ABCG2, ALDH and CD 117 in whole cell population of human omentum fat, subcutaneous fat and bone marrow. The phenotypic characterization through flowcytometry revealed the positive expressions of CD 34, CD 45, CD 133, HLADR, CD 90, CD 105, CD 73, CD 29, CD 13, CD 44, CD 54, CD 31, ALDH and CD 117 in all sources. The similarities between the phenotypic expressions of omentum fat derived stem cells to that of subcutaneous fat and bone marrow substantiates that identification of ultimate source for curative therapeutics is arduous to assess. Nevertheless, these results support the potential therapeutic application of omentum fat derived stem cells.

Electronic supplementary material

The online version of this article (doi:10.1007/s10616-012-9427-4) contains supplementary material, which is available to authorized users.     

低氧对MSCs增殖和分化的影响

间充质干细胞（mesenchymal stemcells, MSCs）是具有自我更新、多向分化和强可塑性的细胞,具有分化为血液、骨、软骨、脂肪、肌肉、表皮、上皮、神经等组织的潜能,受到再生医学研究的关注。目前已有研究表明将MSCs 移植到多种损伤组织中都能改善损伤组织的功能。文章在简要回顾了低氧环境对MSCs增殖和分化的研究内容和有关理论争论基础上重点介绍了缺氧诱导因子（HIF）通路对MSCs 增殖和分化的影响。文章阐述了低氧环境对MSCs向成骨,成软骨,成脂及成神经元方向分化的影响。由于人体组织内生理条件下的氧张力远远小于大气中的氧张力（21%）,采用低氧培养MSCs 的研究方法得出的结论将更加贴近实际MSCs在人体内的增殖、分化情况。因此研究MSCs 在低氧张力环境中增殖、分化的能力将为MSCs 能成功移植到体内并发挥作用提供保障。     

Modification of the radiation injury of hematopoiesis in rats using the gaseous hypoxic mixture GHM-10

In experiments on Wistar rats it was shown that gas hypoxic mixture containing O2 (10%) and N2 (90%) had a radioprotective action with regard to the survival rate for 30 days and to the haemopoietic system status. The application of gas hypoxic mixture reduced the postirradiation cytopenia in the blood and lowered the degree of the bone marrow depletion by the 3d day following irradiation; DMF was 1.25 as determined by total bone marrow cellularity.     

低氧对 MSCs 增殖和分化的影响

摘要： 间充质干细胞(mesenchymal stem cells, MSCs)是具有自我更新、 多向分化和强可塑性的细胞, 具有分化为血液、 骨、 软骨、 脂 肪、 肌肉、 表皮、 上皮、 神经等组织的潜能, 受到再生医学研究的关注。目前已有研究表明将 MSCs 移植到多种损伤组织中都能改 善损伤组织的功能。文章在简要回顾了低氧环境对 MSCs 增殖和分化的研究内容和有关理论争论基础上重点介绍了缺氧诱导因 子 （ HIF ）通路对 MSCs 增殖和分化的影响。文章阐述了低氧环境对 MSCs 向成骨,成软骨,成脂及成神经元方向分化的影响。 由于 人体组织内生理条件下的氧张力远远小于大气中的氧张力 （21% ）, 采用低氧培养 MSCs 的研究方法得出的结论将更加贴近实际 MSCs 在人体内的增殖、分化情况。因此研究 MSCs 在低氧张力环境中增殖、分化的能力将为 MSCs 能成功移植到体内并发挥作 用提供保障。     

Bone marrow fat mobilization in relation to lipid and protein catabolism during prolonged fasting in barn owls

 To assess the role of bone marrow fat in survival during a period of negative energy balance, we investigated the relationship between the time-course of marrow fat mobilization and the metabolic states associated with body fuel utilization during a prolonged fast. In order to mimic the winter fast of the barn owl (Tyto alba), captive birds were subjected to fasts of various durations at 5 °C ambient temperature. Body mass and plasma metabolites were used to determine the metabolic state at the end of fasting. Skeleton composition remained unchanged throughout phase II of fasting, during which the birds essentially rely on lipid fuels. During the following phase III, characterized by an increase in net body protein breakdown, the lipid mass in skeleton marrow decreased sharply by 78%, concomitant with an increase of the bone water content. This marrow fat mobilization occurred in all parts of the skeleton. This observation supports the hypothesis that bone marrow fat is not only involved in local nutrition, but can also be used as a lipid reserve for total energy requirements. However, in contrast to other fat deposits, marrow fat is mobilized only during phase III of the fast, when the last shift from lipid to protein fuel metabolism occurs. Thus, metabolic and/or hormonal changes associated with this transition could be involved in bone marrow fat mobilization. Lastly, our results suggest that the measurement of bone marrow fat can be used as an accurate index of the nutritional status (i.e. phase II or phase III) in barn owls. Accepted: 4 July 1996     

Proliferation and differentiation of bone marrow stromal cells under hypoxic conditions

Low oxygen tension is a potent differentiation inducer of numerous cell types and an effective stimulus of many gene expressions. Here, we described that under 8% O(2), bone marrow stromal cells (MSCs) exhibited proliferative and morphologic changes. The level of differentiated antigen H-2Dd and the number of G(2)/S/M phase cells increased evidently under 8% O(2) condition. Also, the proportion of wide, flattened, and epithelial-like cells (which were alkaline phosphatase staining positive) in MSCs increased significantly. When cultured in adipogenic medium, there was a 5- to 6-fold increase in the number of lipid droplets under hypoxic conditions compared with that in normoxic culture. We also demonstrated the existence of MSC differentiation under hypoxic conditions by electron microscopy. Expression of Oct4 was inhibited under 8% O(2) condition, but after adipocyte differentiation in normoxic culture and hypoxia-mimicking agents cobalt chloride (CoCl(2)) and deferoxamine mesylate (DFX) treatments, Oct4 was still expressed in MSCs. These results indicate hypoxia accelerates MSC differentiation and hypoxia and hypoxia-mimicking agents exert different effects on MSC differentiation.     

Magnetic Resonance Imaging of Changes in Abdominal Compartments in Obese Diabetics during a Low-Calorie Weight-Loss Program

ObjectivesTo investigate changes in the fat content of abdominal compartments and muscle area during weight loss using confounder-adjusted chemical-shift-encoded magnetic resonance imaging (MRI) in overweight diabetics.MethodsTwenty-nine obese diabetics (10/19 men/women, median age: 59.0 years, median body mass index (BMI): 34.0 kg/m²) prospectively joined a standardized 15-week weight-loss program (six weeks of formula diet exclusively, followed by reintroduction of regular food with gradually increasing energy content over nine weeks) over 15 weeks. All subjects underwent a standardized MRI protocol including a confounder-adjusted chemical-shift-encoded MR sequence with water/fat separation before the program as well at the end of the six weeks of formula diet and at the end of the program at 15 weeks. Fat fractions of abdominal organs and vertebral bone marrow as well as volumes of visceral and subcutaneous fat were determined. Furthermore, muscle area was evaluated using the L4/L5 method. Data were compared using the Wilcoxon signed-rank test for paired samples.ResultsMedian BMI decreased significantly from 34.0 kg/m² to 29.9 kg/m² (p < 0.001) at 15 weeks. Liver fat content was normalized (14.2% to 4.1%, p < 0.001) and vertebral bone marrow fat (57.5% to 53.6%, p = 0.018) decreased significantly throughout the program, while fat content of pancreas (9.0%), spleen (0.0%), and psoas muscle (0.0%) did not (p > 0.15). Visceral fat volume (3.2 L to 1.6 L, p < 0.001) and subcutaneous fat diameter (3.0 cm to 2.2 cm, p < 0.001) also decreased significantly. Muscle area declined by 6.8% from 243.9 cm² to 226.8 cm².ConclusionMRI allows noninvasive monitoring of changes in abdominal compartments during weight loss. In overweight diabetics, weight loss leads to fat reduction in abdominal compartments, such as visceral fat, as well as liver fat and vertebral bone marrow fat while pancreas fat remains unchanged.     

Effect of high glucose on extensive culturing of mesenchymal stem cells derived from subcutaneous fat,omentum fat and bone marrow

Frontline research progresses the applicability of bone marrow and adipose tissue in regenerative medicine, but fails to account for the functional improvement of the diseased. The justification for the failure in terms of stem cell survival, proliferation and regeneration is unclear. However, hyperglycemia rising during pathological conditions might be one such stumbling block. The prevailing literature accounts for both detrimental and beneficial effect of high glucose on mesenchymal stem cells (MSCs) leading to perplexity. Thus, this study focuses on the effect of high glucose on mesenchymal stem cells derived from subcutaneous fat, omentum fat and bone marrow in extensive cultures. We provide evidence for the retention of MSC characteristics of all sources with regards to surface marker profiling, proliferation, differentiation and karyotyping when cultured extensively under DMEM‐HG containing glucose concentration of 25 mmol.l^–1. Thus, it can be concluded that hyperglycemia in vivo (11 mmol.l^–1) might not be a barrier for the ineffective functional improvement of transplanted stem cells. Furthermore, we elucidated subcutaneous and omentum fat as better sources of MSCs when compared with bone marrow, thereby making these sources optimal for therapies during hyperglycemic conditions. However, further research is needed to clear the path for efficient stem cell transplantation. Copyright © 2012 John Wiley & Sons, Ltd.     

Bone marrow long label-retaining cells reside in the sinusoidal hypoxic niche

In response to changing signals, quiescent hematopoietic stem cells (HSCs) can be induced to an activated cycling state and provide multi-lineage hematopoietic cells to the whole body via blood vessels. However, the precise localization of quiescent HSCs in bone marrow microenvironment is not fully characterized. Here, we performed whole-mount immunostaining of bone marrow and found that BrdU label-retaining cells (LRCs) definitively reside in the sinusoidal hypoxic zone distant from the “vascular niche”. Although LRCs expressed very low level of a well-known HSC marker, c-kit in normal circumstances, myeloablation by 5-FU treatment caused LRCs to abundantly express c-kit and proliferate actively. These results demonstrate that bone marrow LRCs reside in the sinusoidal hypoxic niche, and function as a regenerative cell pool of HSCs.     

Composition of adipose tissue and marrow fat in humans by 1H NMR at 7 Tesla

Proton NMR spectroscopy at 7 Tesla (7T) was evaluated as a new method to quantify human fat composition noninvasively. In validation experiments, the composition of a known mixture of triolein, tristearin, and trilinolein agreed well with measurements by (1)H NMR spectroscopy. Triglycerides in calf subcutaneous tissue and tibial bone marrow were examined in 20 healthy subjects by (1)H spectroscopy. Ten well-resolved proton resonances from triglycerides were detected using stimulated echo acquisition mode sequence and small voxel ( approximately 0.1 ml), and T(1) and T(2) were measured. Triglyceride composition was not different between calf subcutaneous adipose tissue and tibial marrow for a given subject, and its variation among subjects, as a result of diet and genetic differences, fell in a narrow range. After correction for differential relaxation effects, the marrow fat composition was 29.1 +/- 3.5% saturated, 46.4 +/- 4.8% monounsaturated, and 24.5 +/- 3.1% diunsaturated, compared with adipose fat composition, 27.1 +/- 4.2% saturated, 49.6 +/- 5.7% monounsaturated, and 23.4 +/- 3.9% diunsaturated. Proton spectroscopy at 7T offers a simple, fast, noninvasive, and painless method for obtaining detailed information about lipid composition in humans, and the sensitivity and resolution of the method may facilitate longitudinal monitoring of changes in lipid composition in response to diet, exercise, and disease.     

Vertebral Bone Marrow Fat Is Positively Associated With Visceral Fat and Inversely Associated With IGF‐1 in Obese Women

Recent studies have demonstrated an important physiologic link between bone and fat. Bone and fat cells arise from the same mesenchymal precursor cell within bone marrow, capable of differentiation into adipocytes or osteoblasts. Increased BMI appears to protect against osteoporosis. However, recent studies have suggested detrimental effects of visceral fat on bone health. Increased visceral fat may also be associated with decreased growth hormone (GH) and insulin‐like growth factor 1 (IGF‐1) levels which are important for maintenance of bone homeostasis. The purpose of our study was to assess the relationship between vertebral bone marrow fat and trabecular bone mineral density (BMD), abdominal fat depots, GH and IGF‐1 in premenopausal women with obesity. We studied 47 premenopausal women of various BMI (range: 18–41 kg/m², mean 30 ± 7 kg/m²) who underwent vertebral bone marrow fat measurement with proton magnetic resonance spectroscopy (1H‐MRS), body composition, and trabecular BMD measurement with computed tomography (CT), and GH and IGF‐1 levels. Women with high visceral fat had higher bone marrow fat than women with low visceral fat. There was a positive correlation between bone marrow fat and visceral fat, independent of BMD. There was an inverse association between vertebral bone marrow fat and trabecular BMD. Vertebral bone marrow fat was also inversely associated with IGF‐1, independent of visceral fat. Our study showed that vertebral bone marrow fat is positively associated with visceral fat and inversely associated with IGF‐1 and BMD. This suggests that the detrimental effect of visceral fat on bone health may be mediated in part by IGF‐1 as an important regulator of the fat and bone lineage.     

Romanovsky Staining of Low Viscosity Nitrocellulose Sections of Bone Marrow

In the detailed study of bone marrow, sections of undisturbed marrow are essential in determining the distribution and relative proportion of the various hemopoietic cells and the relative volume occupied by marrow cells, fat, and blood in the various portions of the same bone or of different bones. As a result of trial of a number of fixatives, stains, and embedding technics, the following technic was evolved. It gives the best combination of good differential staining with the least shrinkage and cellular distortion, and is used routinely by the author in the study of bone marrow lesions in rats.     

Histology of the bone marrow antibody response

The histology of the specific and non-specific antibody response in mouse and rat bone marrow was studied after subcutaneous priming and intravenous boosting with horseradish peroxidase (HRP). Cells producing specific antibody against HRP were found only occasionally in the bone marrow after subcutaneous priming. After the intravenous boost injection their number gradually increased. These anti-HRP forming cells were found as single cells, randomly dispersed throughout the bone marrow. Such a random distribution was also found for cytoplasmic (non-specific) immunoglobulin containing cells. At no time point after immunization could lymphoid aggregates or trapping of immune complexes be observed in the bone marrow of either species. On the basis of these observations it is concluded that the bone marrow forms a suitable microenvironment for immigrating antibody-forming cells but does not contribute actively to the induction of the immune response.     

Role of the spleen in the exaggerated polycythemic response to hypoxia in chronic mountain sickness in rats.

In a rat model of chronic mountain sickness, the excessive polycythemic response to hypoxic exposure is associated with profound splenic erythropoiesis. We studied the uptake and distribution of radioactive iron and red blood cell (RBC) morphology in intact and splenectomized rats over a 30-day hypoxic exposure. Retention of (59)Fe in the plasma was correlated with (59)Fe uptake by both spleen and marrow and the appearance of (59)Fe-labeled RBCs in the blood. (59)Fe uptake in both the spleen and the marrow paralleled the production of nucleated RBCs. Splenic (59)Fe uptake was approximately 10% of the total marrow uptake under normoxic conditions but increased to 60% of the total marrow uptake during hypoxic exposure. Peak splenic (59)Fe uptake and splenomegaly occurred at the most intense phase of erythropoiesis and coincided with the rapid appearance of (59)Fe-labeled RBCs in the blood. The bone marrow remains the most important erythropoietic organ under both resting and stimulated states, but inordinate splenic erythropoiesis in this rat strain accounts in large measure for the excessive polycythemia during the development of chronic mountain sickness in chronic hypoxia.     

Disseminated Breast Cancer Cells Acquire a Highly Malignant and Aggressive Metastatic Phenotype during Metastatic Latency in the Bone

Background

Disseminated tumor cells (DTCs) in the bone marrow may exist in a dormant state for extended periods of time, maintaining the ability to proliferate upon activation, engraft at new sites, and form detectable metastases. However, understanding of the behavior and biology of dormant breast cancer cells in the bone marrow niche remains limited, as well as their potential involvement in tumor recurrence and metastasis. Therefore, the purpose of this study was to investigate the tumorigenicity and metastatic potential of dormant disseminated breast cancer cells (prior to activation) in the bone marrow.

Methodology/Principal Findings

Total bone marrow, isolated from mice previously injected with tumorspheres into the mammary fat pad, was injected into the mammary fat pad of NUDE mice. As a negative control, bone marrow isolated from non-injected mice was injected into the mammary fat pad of NUDE mice. The resultant tumors were analyzed by immunohistochemistry for expression of epithelial and mesenchymal markers. Mouse lungs, livers, and kidneys were analyzed by H+E staining to detect metastases. The injection of bone marrow isolated from mice previously injected with tumorspheres into the mammary fat pad, resulted in large tumor formation in the mammary fat pad 2 months post-injection. However, the injection of bone marrow isolated from non-injected mice did not result in tumor formation in the mammary fat pad. The DTC-derived tumors exhibited accelerated development of metastatic lesions within the lung, liver and kidney. The resultant tumors and the majority of metastatic lesions within the lung and liver exhibited a mesenchymal-like phenotype.

Conclusions/Significance

Dormant DTCs within the bone marrow are highly malignant upon injection into the mammary fat pad, with the accelerated development of metastatic lesions within the lung, liver and kidney. These results suggest the acquisition of a more aggressive phenotype of DTCs during metastatic latency within the bone marrow microenvironment.     

Morphometric and hormonal changes during the chimpanzee menstrual cycle

BACKGROUND: Sex steroids affect many peripheral tissue sites in female mammals. Receptors for these hormones have been found in skin, fat, and bone. In women, these tissues can show morphological changes during the menstrual cycle that may be directly related to steroid secretion. METHODS: The present study was done on chimpanzees to document morphometric markers associated with these tissues (anogenital swelling volume, skin fold thickness as indicator of subcutaneous fat, bony diameters of mandible, wrist, and elbow) and to compare them with cyclic patterns of estradiol, progesterone, testosterone, gonadotropins, and prolactin. RESULTS: Swelling volume changed significantly over the menstrual cycle. All other morphometric parameters showed variation without statistical significance. Skin folds were thickest during the luteal phase. Bony diameters displayed similar but less distinctive changes. Testosterone correlated positively with diameter sites, inversely with subcutaneous fat. No relationships with either estradiol or progesterone were found. We assume that subcutaneous fat and morphometric bone parameters exhibit cycle-dependent changes that may be caused by changes in steroid secretion.     

Hypoxia is a major stimulator of osteoclast formation and bone resorption

Hypoxia is known to act as a general stimulator of cells derived from marrow precursors. We investigated the effect of oxygen tension on the formation and function of osteoclasts, the cells responsible for bore resorption, which are of promonocytic origin. Using 7- and 13-day cultures of mouse marrow cells on ivory discs, we found that reducing oxygen tension from the ambient atmospheric level of 20% by increasing the proportion of nitrogen caused progressive increases in the formation of multinucleated osteoclasts and resorption pits. Peak effects occurred in 2% oxygen, where stimulations of resorption up to 21-fold were measured. Significant stimulations of osteoclast formation and resorption were observed even in severely hypoxic cultures gassed with 0.2% oxygen. Short-term cultures of cells disaggregated from rat bones indicated that hypoxia did not alter the resorptive activity of mature osteoclasts, but reduced their survival or adherence. In 3-day organ cultures of mouse calvarial bones, exposure to 2% oxygen resulted in maximal, fivefold stimulation of osteoclast-mediated calcium release, an effect equivalent to that of prostaglandin E(2) (PGE(2)), a reference osteolytic agent. Hypoxia also caused a moderate acidosis in calvarial cultures, presumably as a result of increased anaerobic metabolism; this observation is significant because osteoclast activation is dependent on extracellular acidification. Our experiments reveal a previously-overlooked mechanism of considerable potential importance for the regulation of bone destruction. These findings may help explain the bone loss associated with a wide range of pathological states involving local or systemic hypoxia, and emphasize the importance of the vasculature in bone.     

Effects of hypoxia on the small acetylcholinesterase-positive megakaryocyte precursor in bone marrow of mice

The number of small acetylcholinesterase-positive (SAChE+) cells in the marrow of hypoxic mice was measured. Mice were exposed to 6-7% O2 levels by enclosure in cages covered with dimethyl-silicone rubber membranes for 1-14 days. The mice showed a linear increase in packed cell volumes with time in the hypoxic atmosphere, but platelet counts showed a characteristic biphasic response, i.e., increased platelet counts were observed after 1-3 days of hypoxia, and significantly (P less than 0.05-P less than 0.0005) decreased platelet counts were observed thereafter (6-14 days). The total number of megakaryocytes in the marrow of hypoxic mice decreased significantly (P less than 0.005) with time. In agreement with the data on platelet counts, hypoxia caused the total number of SAChE+ cells in the marrow of mice to be biphasic. At Day 2 there was a significant increase (P less than 0.05) in the total number of SAChE+ cells/mm3 of bone marrow; however, by days 10-14 the numbers had decreased markedly (P less than 0.005). These data indicate that hypoxia decreases platelet production by action on a precursor cell to the SAChE+ cell. The hypoxia-induced thrombocytopenia is probably caused by stem-cell competition between the erythrocytic and megakaryocytic cell lines.