A practical guide to culturing mouse and human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs, frequently also called MSCs) represent a cell population within the bone marrow, a subset of which contains multipotent stem cells. Their primary role is to produce and maintain both bone tissue and bone marrow microenvironment necessary for hematopoiesis. The latter is achieved by secreting a wide variety of different cytokines and growth factors, many of which also have a regulatory role in immune processes. BMSCs have recently been introduced into the field of immunobiology after their successful clinical use in GVHD was reported in 2004. Since then, numerous studies confirmed and expanded the knowledge on the immunosuppressive potential of BMSCs in various in vitro and in vivo models. Although the immunomodulatory capacity of BMSCs is well established, there are still many unanswered questions regarding the cytokines, chemokines, receptors, and molecular pathways that play a role in this effect. To study these cells and answer many of the questions, researchers must be able to reliably and reproducibly isolate, culture, and use these cells. Below a practical guide on how to culture and characterize mouse and human BMSCs, which can then be applied in various in vitro and in vivo assays, is provided.

Bone marrow stromal cells inhibit mast cell function via a COX2-dependent mechanism.

BACKGROUND: Mast cells (MCs) have a central role in the induction of allergic inflammation, such as seen in asthma, and contribute to the severity of certain autoimmune diseases, such as rheumatoid arthritis. The MC thus represents an important inflammatory cell, and one which has resisted therapeutic attempts to alter its role in disease. OBJECTIVE: Because bone marrow-derived stromal cells (BMSC, also known as mesenchymal stem cells or MSCs) have been reported to alter allergic inflammation in vivo, we chose to study the interaction between mouse BMSC and mouse bone marrow-derived MCs. METHODS: MC degranulation, cytokine production and chemotaxis were evaluated in vitro following co-culture with BMSCs either in cell contact or a transwell. In addition, MC degranulation was assessed in vivo following administration of BMSCs in a model of passive cutaneous anaphylaxis and a peritoneal degranulation assay. Mechanisms of MC suppression by BMSCs were determined through use of inhibitors or antibodies to COX1, COX2, nitric oxide, indoleamine 2, 3-dioxygenase, EP1-4 receptors, TGF-ß and IL-10. Lastly, we utilized either BMSCs or MCs deficient in COX1, COX2 or EP1-4 receptors to confirm the mechanisms of inhibition of MC function by BMSCs. RESULTS: We discovered that BMSCs will effectively suppress specific MC functions in vitro as well as in vivo. When MCs are cocultured with BMSCs to allow cell-to-cell contact, BMSCs suppressed MC degranulation, pro-inflammatory cytokine production, chemokinesis and chemotaxis. Similarly, MC degranulation within mouse skin or the peritoneal cavity was suppressed following in vivo administration of BMSCs. Further, we found that these inhibitory effects were dependent on up-regulation of COX2 in BMSCs; and were facilitated through the activation of EP4 receptors on MCs. CONCLUSION AND CLINICAL RELEVANCE: These observations support the concept that BMSCs have the ability to suppress MC activation and therefore could be the basis for a novel cell based therapeutic approach in the treatment of MC driven inflammatory diseases.

Unexpected roles for bone marrow stromal cells (or MSCs): a real promise for cellular, but not replacement, therapy.

Adult and embryonic stem cells have drawn a lot of attention in the last decade as new tools in regenerative medicine. A variety of such cells have been discovered and put forward as candidates for use in cell replacement therapy. Investigators hope that some, if not all, of our organs can be replaced or restored to function; that new livers, kidneys, and brain cells can be produced. Many reviews have already been written about stem cells and their potential use in regenerating tissues. In this study, we would like to call attention to a different application of a special group of adult stem cells, the stromal cells in the bone marrow (also called mesenchymal stem cells or MSCs). These cells have been discovered to modulate immune function. They can easily be expanded in culture and surprisingly, they also seem not to be immunogenic. Thus, they can be removed from donors, expanded, stored in freezers, and used as allogeneic transplants in a variety of diseases in everyday medicine.

Dispersed donor salivary gland cells are widely distributed in the recipient gland when infused up the ductal tree.

The salivary glands often are severely and permanently damaged by therapeutic irradiation for cancer of the head and neck. The markedly reduced quantity and quality of saliva results in greatly increased susceptibility to dental caries and infection of the oral mucosa and alveolar bone. Recently, subcapsular injection of cultured mouse salivary gland cells has achieved a significant degree of regeneration in a previously irradiated mouse salivary gland; however, the recovery was limited to one lobule. We describe here a method for delivering donor rat salivary gland cells via the main duct that distributes several thousand cells throughout the recipient rat's salivary gland. The donated cells exhibited the cytodifferentiation of the structures in which they lodged, i.e., acini, granular convoluted tubules, and the several types of ducts. This method may facilitate the simultaneous functional recovery of almost all of the lobules of irradiated rat salivary glands.

Transforming growth factor alpha induces angiogenesis and neurogenesis following stroke.

The cytokine transforming growth factor alpha (TGF alpha) has proangiogenic and proneurogenic effects and can potentially reduce infarct volumes. Therefore, we administered TGF alpha or vehicle directly into the area surrounding the infarct in female mice that received gender-mismatched bone marrow transplants from green fluorescent protein (GFP)-expressing males prior to undergoing permanent middle cerebral artery occlusion. Newborn cells were tracked with bromodeoxyuridine (BrdU) labeling and immunohistochemistry at 90 days after stroke onset. We also studied the ingress of bone marrow-derived cells into the ischemic brain to determine whether such cells contribute to angiogenesis or neurogenesis. Infarct volumes were measured at 90 days poststroke. The results show that TGF alpha led to significant increments in the number of newborn neurons and glia in the ischemic hemisphere. TGF alpha also led to significant increments in the number of bone marrow-derived cells entering into the ischemic hemisphere. Most of these cells did not label with BrdU and represented endothelial cells that incorporated into blood vessels in the infarct border zone. Our results also show that infarct size was significantly reduced in animals treated with TGF alpha compared with controls. These results suggest that TGF alpha can induce angiogenesis, neurogenesis and neuroprotection after stroke. At least part of the pro-angiogenic effect appears to be secondary to the incorporation of bone marrow-derived endothelial cells into blood vessels in the infarct border zone.

Cells from bone marrow that evolve into oral tissues and their clinical applications.

There are two major well-characterized populations of post-natal (adult) stem cells in bone marrow: hematopoietic stem cells which give rise to blood cells of all lineages, and mesenchymal stem cells which give rise to osteoblasts, adipocytes, and fibroblasts. For the past 50 years, strict rules were taught governing developmental biology. However, recently, numerous studies have emerged from researchers in different fields suggesting the unthinkable--that stem cells isolated from a variety of organs are capable of ignoring their cell lineage boundaries and exhibiting more plasticity in their fates. Plasticity is defined as the ability of post-natal (tissue-specific adult) stem cells to differentiate into mature and functional cells of the same or of a different germ layer of origin. There are reports that bone marrow stem cells can evolve into cells of all dermal lineages, such as hepatocytes, skeletal myocytes, cardiomyocytes, neural, endothelial, epithelial, and even endocrine cells. These findings promise significant therapeutic implications for regenerative medicine. This article will review recent reports of bone marrow cells that have the ability to evolve or differentiate into oral and craniofacial tissues, such as the periodontal ligament, alveolar bone, condyle, tooth, bone around dental and facial implants, and oral mucosa.

Liver alcohol dehydrogenase is degraded by the ubiquitin-proteasome pathway.

Dihydrotestosterone (DHT) decreases rat liver alcohol dehydrogenase (ADH) due principally to an increased rate of degradation of the enzyme. The pathway of degradation of ADH was investigated. Exposure of hepatocytes in culture to lactacystin or to MG132, which are inhibitors of the ubiquitin-proteasome pathway of protein degradation, resulted in higher ADH. Furthermore, both lactacystin and MG132 prevented the decrease in ADH caused by DHT. By contrast, the lysosomal proteolytic inhibitors 3-methyladenine and leupeptin as well as inhibitors of the calcium-activated neutral protease calpain system had no effect on ADH in the absence or presence of DHT. ADH isolated by immunoprecipitation from hepatocytes exposed to DHT reacted specifically with anti-ubiquitin antibody. Ubiquitinated ADH was also demonstrated in hepatocytes exposed to MG132. The combination of DHT and MG132 resulted in more ubiquitinated ADH than exposure to either compound alone. These results suggest that the ubiquitin-proteasome pathway plays a role in the degradation of ADH and in the enhanced degradation of this enzyme by DHT.

Sex difference in alcohol-related organ injury.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Nobuhiro Sato and Kai O. Lindros. The presentations were (1) Sex differences in ethanol pharmacokinetics, by E. Baraona; (2) Estrogen regulates the sensitivity to endotoxin in hepatic Kupffer cells, by K. Ikejima; (3) Sex difference in alcohol-related organ injury, by E. Mezey; (4) Aggravated ethanol-induced liver injury in female rats: Protection by the antiestrogen toremifene, by Harri A. Järveläinen; and (5) Alcohol metabolism in Asian subjects: Sex differences and flushing response, by V. A. Ramchandani.

Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow.

Bone marrow stem cells give rise to a variety of hematopoietic lineages and repopulate the blood throughout adult life. We show that, in a strain of mice incapable of developing cells of the myeloid and lymphoid lineages, transplanted adult bone marrow cells migrated into the brain and differentiated into cells that expressed neuron-specific antigens. These findings raise the possibility that bone marrow-derived cells may provide an alternative source of neurons in patients with neurodegenerative diseases or central nervous system injury.

Distribution of parathyroid hormone-2 receptor-like immunoreactivity and messenger RNA in the rat nervous system.

The parathyroid hormone-2 receptor is a member of the secretin family of guanine nucleotide-binding protein-coupled receptors. The human parathyroid hormone-2 receptor is activated by parathyroid hormone and a recently purified hypothalamic polypeptide, tubero-infundibular peptide of 39 residues, while the rat parathyroid hormone-2 receptor is poorly activated by parathyroid hormone and is potently activated by tubero-infundibular peptide of 39 residues. In order to provide a foundation for studies on the physiological role of the parathyroid hormone-2 receptor and tubero-infundibular peptide of 39 residues, we investigated the cellular distribution of the parathyroid hormone-2 receptor in the rat CNS using both immunohistochemistry and in situ hybridization histochemistry. The receptor is found in discrete groups of neurons in many regions. It is present in scattered small cells throughout the cerebral cortex, in small and medium-sized cells in the striatum, and is quite abundant in the septum and the midline thalamic nuclei. Its expression is high in the hypothalamus, particularly in the periventricular and arcuate nuclei. Fibers and terminals in the external zone of the median eminence, and in the superficial layers of the caudal spinal trigeminal tract and the spinal cord dorsal horn, are strongly and dramatically labeled by a parathyroid hormone-2 receptor-selective antibody. The localization of parathyroid hormone-2 receptor suggests a role in the regulation of pituitary hormone secretion, sensory information processing and homeostatic regulation.

Bone marrow: a possible alternative source of cells in the adult nervous system.

There is increasing evidence that stem cell populations can undergo a transition between mesodermal and neural ectodermal cell fates. Bone marrow-derived cells have been shown to be extremely versatile: they can become brain and liver cells and muscle, while other mesodermal derived cells have been shown to migrate into the brain and differentiate into neurons. Moreover, under the appropriate conditions, neural stem cells can differentiate into hematopoietic and muscle cell fates. It is now well established that newly differentiated cell types are continuously generated from immature stem cells throughout development and in adult mammals, including humans. This review addresses the contribution that bone marrow-derived stem cells may play during neurogenesis. We transplanted male bone marrow into female recipients to track and characterize the Y chromosome containing cells in the CNS (central nervous system) of mice.

Alpha-synuclein immunoreactivity of huntingtin polyglutamine aggregates in striatum and cortex of Huntington's disease patients and transgenic mouse models.

Polyglutamine expansions in proteins are implicated in at least eight inherited neurodegenerative disorders, including Huntington's disease. These mutant proteins can form aggregates within the nucleus and processes of neurons possibly due to misfolding of the proteins. Polyglutamine aggregates are ubiquitinated and sequester molecular chaperone proteins and proteasome components. To investigate other protein components of polyglutamine aggregates, cerebral cortex and striata from patients with Huntington's disease and full-length cDNA transgenic mouse models for this disease were examined immunohistochemically for alpha-synuclein reactivity. Our findings demonstrate that alpha-synuclein can be used as a marker for huntingtin polyglutamine aggregates in both human and mice. Moreover in the HD transgenic mice, the intensity of immunoreactivity increases with age. The significance of recruitment of alpha-synuclein into huntingtin aggregates and its translocation away from the synapses remains to be determined. We propose that aberrant interaction of mutant huntingtin with other proteins, including alpha-synuclein, may influence disease progression.

Neuroserpin is expressed in the pituitary and adrenal glands and induces the extension of neurite-like processes in AtT-20 cells.

Two cDNAs encoding the serine protease inhibitor (serpin) neuroserpin were cloned from a rat pituitary cDNA library (rNS-1, 2922 bp; rNS-2, 1599 bp). In situ hybridization histochemistry showed neuroserpin transcripts in the intermediate, anterior and posterior lobes of the pituitary gland and medullary cells in the adrenal gland. Expression of rNS-1 mRNA was restricted to selected cells in the pituitary gland. Analysis of purified secretory-granule fractions from pituitary and adrenal tissues indicated that neuroserpin was found in dense-cored secretory granules. This result suggested that endocrine neuroserpin may regulate intragranular proteases or inhibit enzymes following regulated secretion. To investigate the function of neuroserpin in endocrine tissues we established stable anterior pituitary AtT-20 cell lines expressing neuroserpin. Cells with increased levels of neuroserpin responded by extending neurite-like processes. Extracellular proteolysis by serine protease plasminogen activators has been suggested to regulate neurite outgrowth. As neuroserpin inhibits tissue plasminogen activator (tPA) in vitro, we measured plasminogen-activator levels. Zymographic analysis indicated that AtT-20 cells synthesized and secreted a plasminogen activator identical in size to tPA. A higher-molecular-mass tPA-neuroserpin complex was also observed in AtT-20-cell conditioned culture medium. tPA levels were similar in parent AtT-20 cells and a stable cell line with increased levels of neuroserpin. There was no accumulation of a tPA-neuroserpin complex. Together these results identify endocrine cells as an important source of neuroserpin. Moreover they suggest that neuroserpin is released from dense-cored secretory granules to regulate cell-extracellular matrix interactions through a mechanism that may not directly involve tPA.

Colocalization of somatostatin receptor sst5 and insulin in rat pancreatic beta-cells.

Somatostatin, also known as somatotropin release-inhibiting factor (SRIF), is secreted by pancreatic delta-cells and inhibits the secretion of both insulin and glucagon. SRIF initiates its actions by binding to a family of six G protein-coupled receptors (sst1, -2A, -2B, -3, -4, and -5) encoded by five genes. Messenger RNA for both sst2 and sst5 have been reported in the rat pancreas, and the sst2A receptor protein has been localized to rat pancreatic alpha and pancreatic polypeptide-secreting cells in the islets as well as to pancreatic acinar cells. In this study we have used double immunostaining to show that the sst5 protein is expressed exclusively in the beta-cells of rat pancreatic islets and localizes with insulin-secreting alpha-cells. The sst5 receptor is not colocalized with sst2A. Thus, in the rat SRIF inhibits pancreatic insulin and glucagon secretion via different sst receptor subtypes.

A transient increase in c-myc precedes the transdifferentiation of hepatic stellate cells to myofibroblast-like cells.

AIMS/BACKGROUND: Liver stellate cells are transdifferentiated to collagen-producing myofibroblast-like cells in vivo during liver injury or when placed in culture. The purpose of this study was to determine the presence of retinoids and the expression of the immediate early genes as they relate to the transdifferentiation of liver stellate cells in culture. METHODS: Rat liver stellate cells were studied immediately after isolation or sequentially after culture for varying periods of time. RNA was isolated and specific messages were determined by RT-PCR. Cells were also isolated for determination of retinoid autofluorescence and immunofluorescent staining with specific antibodies by laser confocal microscopy. RESULTS: c-fos message and immunoprotein were high in the freshly isolated cells prior to culture, while c-myc expression increased markedly after one day of culture. Both c-fos and c-myc gene expression decreased prior to the transdifferentiation of the cells to myofibroblast-like cells and to the increase in alpha 1(I) and alpha 2(I) collagen messages and collagen production. The presence of retinoid autofluorescence and retinoic acid receptor (RAR-alpha and RAR-beta) messages and RAR-beta immunoprotein persisted during initial transdifferentiation of the stellate cells. CONCLUSIONS: This study shows a high initial level of c-fos expression and a transient increase in c-myc expression followed by a decrease to lower levels prior to transdifferentiation and collagen production by stellate cells. A total loss of retinoid autofluorescence or a decrease in RAR-alpha or RAR-beta are not required for initial transdifferentiation of stellate cells or collagen production.

Plasma metanephrines are markers of pheochromocytoma produced by catechol-O-methyltransferase within tumors.

This study examined whether the high sensitivity of plasma free metanephrines for diagnosis of pheochromocytoma may result from production of free metanephrines within tumors. Presence in pheochromocytomas of catechol-O-methyltransferase (COMT), the enzyme responsible for conversion of catecholamines to metanephrines, was confirmed by Western blot analysis, enzyme assay, and immunohistochemistry. Western blot analysis and enzyme assay indicated that membrane-bound and not soluble COMT was the predominant form of the enzyme in pheochromocytoma. Immunohistochemistry revealed colocalization of COMT in the same chromaffin cells where catecholamines are translocated into storage vesicles by the vesicular monoamine transporter. Levels of free metanephrines in pheochromocytoma over 10,000 times higher than plasma concentrations in the same patients before removal of tumors indicated production of metanephrines within tumors. Comparisons of the production of metanephrines in patients with pheochromocytoma with production from catecholamines released or infused into the circulation indicated that more than 93% of the consistently elevated levels of circulating free metanephrines in patients with pheochromocytoma are derived from metabolism before and not after release of catecholamines into the circulation. The data indicate that the elevated plasma levels of free metanephrines in patients with pheochromocytoma are derived from catecholamines produced and metabolized within tumors. Some tumors do not secrete catecholamines, but all appear to metabolize catecholamines to free metanephrines, thus explaining the better sensitivity of plasma free metanephrines over other tests for diagnosis of pheochromocytoma.

Cell specific expression of the sst2A and sst5 somatostatin receptors in the rat anterior pituitary.

Somatostatin (SRIF), originally described as a hypothalamic hormone that inhibits the release of growth hormone was subsequently shown to inhibit the secretion of multiple pituitary hormones. Five genes encoding six different SRIF receptors (sst1, 2A, 2B, 3, 4 and 5) have been cloned and mRNAs for all five are expressed in the anterior pituitary. We used double immunostaining to determine which cells in the anterior pituitary bear sst2A and sst5 receptors. Our results show that these two receptors are widely distributed in the pituitary gland and are both present in a large percentage of GH cells. In addition, sst5 occurs in a small population of corticotrophs and a large percentage of lactotrophs whereas sst2A is found in only a few lactotrophs but a large number of corticotrophs. The sst2A receptor is also expressed in about a third of the gonadotrophs and thyrotrophs. Interestingly, sst2A and sst5 receptors colocalize in a small percentage of cells, most likely somatotrophs demonstrating that the same cells can contain multiple sst receptor subtypes. These results indicate that sst subtype specific analogs are likely to be useful for the selective regulation of individual pituitary hormones.