Human Multipotent Mesenchymal Stromal Cells from Distinct Sources Show Different In Vivo Potential to Differentiate into Muscle Cells When Injected in Dystrophic Mice

Limb-girdle muscular dystrophies are a heterogeneous group of disorders characterized by progressive degeneration of skeletal muscle caused by the absence or deficiency of muscle proteins. The murine model of Limb-Girdle Muscular Dystrophy 2B, the SJL mice, carries a deletion in the dysferlin gene. Functionally, this mouse model shows discrete muscle weakness, starting at the age of 4-6 weeks. The possibility to restore the expression of the defective protein and improve muscular performance by cell therapy is a promising approach for the future treatment of progressive muscular dystrophies (PMD). We and others have recently shown that human adipose multipotent mesenchymal stromal cells (hASCs) can differentiate into skeletal muscle when in contact with dystrophic muscle cells in vitro and in vivo. Umbilical cord tissue and adipose tissue are known rich sources of multipotent mesenchymal stromal cells (MSCs), widely used for cell-based therapy studies. The main objective of the present study is to evaluate if MSCs from these two different sources have the same potential to reach and differentiate in muscle cells in vivo or if this capability is influenced by the niche from where they were obtained. In order to address this question we injected human derived umbilical cord tissue MSCs (hUCT MSCs) into the caudal vein of SJL mice with the same protocol previously used for hASCs; we evaluated the ability of these cells to engraft into recipient dystrophic muscle after systemic delivery, to express human muscle proteins in the dystrophic host and their effect in functional performance. These results are of great interest for future therapeutic application.

Chlorella vulgaris up-modulation of myelossupression induced by lead: The role of stromal cells

In this study, Chlorella vulgaris (CV) was examined for its chelating effects on the ability of bone marrow stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice, using the long-term bone marrow culture (LTBMC). In addition, the levels of interleukin (IL)-6, an important hematopoietic stimulator, as well as the numbers of adherent and non-adherent cells were also investigated. Mice were gavage treated daily with a single 50 mg/kg dose of CV for 10 days, concomitant to continuous offering of 1300 ppm lead acetate in drinking water. We found that CV up-modulates the reduced ability of stromal cell layer to display myeloid progenitor cells in vitro in lead-exposed mice and restores both the reduced number of non-adherent cells and the ability of stromal cells from these mice to produce IL-6. Monitoring of lead poisoning demonstrated that CV treatment significantly reduced lead levels in blood and tissues, completely restored the normal hepatic ALA levels, decreased the abnormally high plasma ALA and partly recovered the liver capacity to produce porphyrins. These findings provide evidence for a beneficial use of CV for combination or alternative chelating therapy to protect the host from the damage induced by lead poisoning. (C) 2008 Elsevier Ltd. All rights reserved.

Multipotent stem cells from umbilical cord: Cord is richer than blood!

The identification of mesenchymal stem cell ( MSC) sources that are easily obtainable is of utmost importance. Several studies have shown that MSCs could be isolated from umbilical cord (UC) units. However, the presence of MSCs in umbilical cord blood (UCB) is controversial. A possible explanation for the low efficiency of MSCs from UCB is the use of different culture conditions by independent studies. Here, we compared the efficiency in obtaining MSCs from unrelated paired UCB and UC samples harvested from the same donors. Samples were processed simultaneously, under the same culture conditions. Although MSCs from blood were obtained from only 1 of the 10 samples, we were able to isolate large amounts of multi-potent MSCs from all UC samples, which were able to originate different cell lineages. Since the routine procedure in UC banks has been to store the blood and discard other tissues, such as the cord and/or placenta, we believe our results are of immediate clinical value. Furthermore, the possibility of originating different cell lines from the UC of neonates born with genetic defects may provide new cellular research models for understanding human malformations and genetic disorders, as well as the possibility of testing the effects of different therapeutic drugs.

Isolation, Characterization, and Differentiation Potential of Canine Adipose-Derived Stem Cells

Adipose tissue may represent a potential source of adult stem cells for tissue engineering applications in veterinary medicine. It can be obtained in large quantities, under local anesthesia, and with minimal discomfort. In this study, canine adipose tissue was obtained by biopsy from subcutaneous adipose tissue or by suction-assisted lipectomy (i.e., liposuction). Adipose tissue was processed to obtain a fibroblast-like population of cells similar to human adipose-derived stem cells (hASCs). These canine adipose-derived stem cells (cASCs) 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 cASCs are of mesodermal or mesenchymal origin. cASCs are able to differentiate in vitro into adipogenic, chondrogenic, myogenic, and osteogenic cells in the presence of lineage-specific induction factors. In conclusion, like human lipoaspirate, canine adipose tissue may also contain multipotent cells and represent an important stem cell source both for veterinary cell therapy as well as preclinical studies.

Gene Expression Profile of Mesenchymal Stem Cells from Paired Umbilical Cord Units: Cord is Different from Blood

Mesenchymal stem cells (MSC) are multipotent cells which can be obtained from several adult and fetal tissues including human umbilical cord units. We have recently shown that umbilical cord tissue (UC) is richer in MSC than umbilical cord blood (UCB) but their origin and characteristics in blood as compared to the cord remains unknown. Here we compared, for the first time, the exonic protein-coding and intronic noncoding RNA (ncRNA) expression profiles of MSC from match-paired UC and UCB samples, harvested from the same donors, processed simultaneously and under the same culture conditions. The patterns of intronic ncRNA expression in MSC from UC and UCB paired units were highly similar, indicative of their common donor origin. The respective exonic protein-coding transcript expression profiles, however, were significantly different. Hierarchical clustering based on protein-coding expression similarities grouped MSC according to their tissue location rather than original donor. Genes related to systems development, osteogenesis and immune system were expressed at higher levels in UCB, whereas genes related to cell adhesion, morphogenesis, secretion, angiogenesis and neurogenesis were more expressed in UC cells. These molecular differences verified in tissue-specific MSC gene expression may reflect functional activities influenced by distinct niches and should be considered when developing clinical protocols involving MSC from different sources. In addition, these findings reinforce our previous suggestion on the importance of banking the whole umbilical cord unit for research or future therapeutic use.

Bone marrow mononuclear cells attenuate fibrosis development after severe acute kidney injury

One of the early phases that lead to fibrosis progression is inflammation. Once this stage is resolved, fibrosis might be prevented. Bone marrow mononuclear cells (BMMCs) are emerging as a new therapy for several pathologies, including autoimmune diseases, because they enact immunosuppression. In this study we aimed to evaluate the role of BMMC administration in a model of kidney fibrosis induced by an acute injury. C57Bl6 mice were subjected to unilateral severe ischemia by clamping the left renal pedicle for 1 h. BMMCs were isolated from femurs and tibia, and after 6 h of reperfusion, 1 x 10(6) cells were administrated intraperitoneally. At 24 h after surgery, treated animals showed a significant decrease in creatinine and urea levels when compared with untreated animals. Different administration routes were tested. Moreover, interferon (IFN) receptor knockout BMMCs were used, as this receptor is necessary for BMMC activation. Labeled BMMCs were found in ischemic kidney on FACS analysis. This improved outcome was associated with modulation of inflammation in the kidney and systemic modulation, as determined by cytokine expression profiling. Despite non-amelioration of functional parameters, kidney mRNA expression of interleukin (IL)-6 at 6 weeks was lower in BMMC-treated animals, as were levels of collagen 1, connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-beta) and vimentin. Protective molecules, such as IL-10, heme oxygenase 1 (HO-1) and bone morphogenetic 7 (BMP-7), were increased in treated animals after 6 weeks. Moreover, Masson and Picrosirius red staining analyses showed less fibrotic areas in the kidneys of treated animals. Thus, early modulation of inflammation by BMMCs after an ischemic injury leads to reduced fibrosis through modulation of early inflammation.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

Stem cell Researches in Brazil: Present and Future Challenges

A bill allowing researches with human embryonic stem cells has been approved by the Brazilian Congress, originally in 2005 and definitively by the Supreme Court in 2008. However, several years before, investigations in Brazil with adult stem cells in vitro in animal models as well as clinical trials, were started and are currently underway. Here, we will summarize the main findings and the challenges of going from bench to bed, focusing on heart, diabetes, cancer, craniofacial, and neuromuscular disorders. We also call attention to the importance of publishing negative results on experimental trials in scientific journals and websites. They are of great value to investigators in the field and may avoid the repeating of unsuccessful experiments. In addition, they could be referred to patients seeking information, aiming to protect them against financial and psychological harm.

Early modulation of inflammation by mesenchymal stem cell after acute kidney injury

Therapy with stem cells has showed to be promising for acute kidney injury (AKI), although how it works is still controversial. Modulation of the inflammatory response is one possible mechanism. Most of published data relies on early time and whether the protection is still maintained after that is not known. Here, we analyzed whether immune modulation continues after 24 h of reperfusion. MSC were obtained from male Wistar rats. After 3-5 passages, cells were screened for CD73, CD90, CD44, CD45, CD29 and CD 31. In addition, MSC were submitted to differentiation in adipocyte and in osteocyte. AKI was induced by bilaterally clamping of renal pedicles for 60 min. Six hours after injury, MSC (2 x 105 cells) were administered intravenously. MSC-treated animals presented the lowest serum creatinine compared to non-treated animals (24 h: 1.3 +/- 0.21 vs. 3.23 +/- 0.89 mg/dl, p<0.05). The improvement in renal function was followed by a lower expression of IL-1b, IL-6 and TNF-alpha and higher expression of IL-4 and IL-10. However, 48 h after reperfusion, this cytokine profile has changed. The decrease in Th1 cytokines was less evident and IL-6 was markedly up regulated. PCNA analysis showed that regeneration occurs faster in kidney tissues of MSC-treated animals than in controls at 24 h. And also ratio of Bcl-2/Bad was higher at treated animals after 24 and 48 h. Our data demonstrated that the immunomodulatory effects of MSC occur at very early time point, changing the inflammation profile toward a Th2 profile. (C) 2009 Elsevier B.V. All rights reserved.

iNOS-derived Nitric Oxide Modulates Infection-stimulated Bone Loss

Nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) plays an important role in host defense, as well as in inflammation-induced tissue lesions. Here we evaluated the role of NO in bone loss in bacterial infection-induced apical periodontitis by using iNOS-deficient mice (iNOS(-/-)). The iNOS(-/-) mice developed greater inflammatory cell recruitment and osteolytic lesions than WT mice. Moreover, tartrate-resistant acid-phosphatase-positive (TRAP(+)) osteoclasts were significantly more numerous in iNOS-/- mice. Furthermore, the increased bone resorption in iNOS(-/-) mice also correlated with the increased expression of receptor activator NF-kappaB (RANK), stromal-cell-derived factor-1 alpha (SDF-1 alpha/CXCL12), and reduced expression of osteoprotegerin (OPG). These results show that NO deficiency was associated with an imbalance of bone-resorption-modulating factors, leading to severe infection-stimulated bone loss.

Stromal-derived factor-1 alpha (CXCL12) levels increase in periodontal disease

Background: The CXC chemokine receptor 4 (CXCR4) and its ligand, stromal cell-derived factor-1 (SDF-1 alpha or CXC chemokine ligand 12) are involved in the trafficking of leukocytes into and out of extravascular tissues. The purpose of this study was to determine whether SDF-1 alpha secreted by host cells plays a role in recruiting inflammatory cells into the periodontia during local inflammation. Methods: SDF-1 alpha levels were determined by enzyme-linked immunosorbent assay in gingival crevicular fluid (GCF) of 24 individuals with periodontitis versus healthy individuals in tissue biopsies and in a preclinical rat model of Porphyromonas gingivalis lipopolysaccharide-induced experimental bone loss. Neutrophil chemotaxis assays were also used to evaluate whether SDF-1 alpha plays a role in the recruitment of host cells at periodontal lesions. Results: Subjects with periodontal disease had higher levels of SDF-1 alpha in their GCF compared to healthy subjects. Subjects with periodontal disease who underwent mechanical therapy demonstrated decreased levels of SDF-1 alpha. Immunohistologic staining showed that SDF-1 alpha and CXCR4 levels were elevated in samples obtained from periodontally compromised individuals. Similar results were observed in the rodent model. Neutrophil migration was enhanced in the presence of SDF-1 alpha, mimicking immune cell migration in periodontal lesions. Conclusions: SDF-1 alpha may be involved in the immune defense pathway activated during periodontal disease. Upon the development of diseased tissues, SDF-1 alpha levels increase and may recruit host defensive cells into sites of inflammation. These studies suggest that SDF-1 alpha may be a useful biomarker for the identification of periodontal disease progression.

Increased Activation of Stromal Interaction Molecule-1/Orai-1 in Aorta From Hypertensive Rats A Novel Insight Into Vascular Dysfunction

Disturbances in the regulation of cytosolic calcium (Ca(2+)) concentration play a key role in the vascular dysfunction associated with arterial hypertension. Stromal interaction molecules (STIMs) and Orai proteins represent a novel mechanism to control store-operated Ca(2+) entry. Although STIMs act as Ca(2+) sensors for the intracellular Ca(2+) stores, Orai is the putative pore-forming component of Ca(2+) release-activated Ca(2+) channels at the plasma membrane. We hypothesized that augmented activation of Ca(2+) release-activated Ca(2+)/Orai-1, through enhanced activity of STIM-1, plays a role in increased basal tonus and vascular reactivity in hypertensive animals. Endothelium-denuded aortic rings from Wistar-Kyoto and stroke-prone spontaneously hypertensive rats were used to evaluate contractions because of Ca(2+) influx. Depletion of intracellular Ca(2+) stores, which induces Ca(2+) release-activated Ca(2+) activation, was performed by placing arteries in Ca(2+) free-EGTA buffer. The addition of the Ca(2+) regular buffer produced greater contractions in aortas from stroke-prone spontaneously hypertensive rats versus Wistar-Kyoto rats. Thapsigargin (10 mu mol/L), an inhibitor of the sarcoplasmic reticulum Ca(2+) ATPase, further increased these contractions, especially in stroke-prone spontaneously hypertensive rat aorta. Addition of the Ca(2+) release-activated Ca(2+) channel inhibitors 2-aminoethoxydiphenyl borate (100 mu mol/L) or gadolinium (100 mu mol/L), as well as neutralizing antibodies to STIM-1 or Orai-1, abolished thapsigargin-increased contraction and the differences in spontaneous tone between the groups. Expression of Orai-1 and STIM-1 proteins was increased in aorta from stroke-prone spontaneously hypertensive rats when compared with Wistar-Kyoto rats. These results support the hypothesis that both Orai-1 and STIM-1 contribute to abnormal vascular function in hypertension. Augmented activation of STIM-1/Orai-1 may represent the mechanism that leads to impaired control of intracellular Ca(2+) levels in hypertension. (Hypertension. 2009; 53[part 2]: 409-416.)