Phenotypic heterogeneity of human cardiac stem cell clones and cardiosphere-forming cells

Although cardiac stem cells have been isolated based on stem cell surface markers, no single marker is stem cell-specific. Clonogenicity is a defining functional property of stemness. We therefore analyzed cardiac cell clones derived from human hearts.Methods: Clonogenic cells were derived from adult human atrial samples. Cells were either cultured in the absence of an initial marker selection or, in separate experiments, they were initially selected for c-kit (CD117), CD31 or CD164 by magnetic immunobeads, or for high aldehyde dehydrogenase activity (ALDH) by FACS. High ALDH activity has been linked to stem/progenitor cells in several tissues. Surface marker analysis was performed by flow cytometry. Cultured cells were also exposed to different factors that modulate cell differentiation, including Dikkopf-1, Noggin, and Wnt-5.Results: Clonogenic cells mainly showed fibroblast-like morphology, ability to grow for more than 30 passages in vitro, and a heterogeneous marker profile even in clones derived from the same cardiac sample. The predominant phenotype was positive for CD13, CD29, CD31, CD44, CD54, CD105 and CD146, but negative for CD10, CD11b, CD14, CD15, CD34, CD38, CD45, CD56, CD106, CD117, CD123, CD133, CD135 and CD271, primarily consistent with endothelial/vascular progenitor cells. However, a minority of clones showed a different profile characterized by expression of CD90, CD106 and CD318, but not CD31 and CD146, consistent with mesenchymal stem/progenitor cells. When initial cell selection was performed, both phenotypes were observed, similarly to unselected cells, irrespective of the selection marker used. Of note, CD117+ sorted cell clones were CD117-negative in culture. Regardless of the immunophenotype, several clones were able to form spheric cell aggregates (cardiospheres), a distinct stem cell property. Dikkopf-1 induced marked CD15 and CD106 upregulation, consistent with stromal differentiation; this effect was prevented by Noggin.Conclusions: The adult human heart contains clonogenic stem/progenitor cells that can be expanded for many passages and form cardiospheres. The surface marker profile of these cells is heterogeneous, consistent with a majority of clones being comprised of endothelial or vascular progenitor cells and a minority of clones consisting of mesenchymal stem/progenitor cells. Dikkopf-1 and Noggin showed opposing effects on stromal differentiation of human cardiac cell clones.

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration.

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.

Magnetic resonance imaging overestimates ferumoxide-labeled stem cell survival after transplantation in the heart.

BACKGROUND: Stem cell labeling with iron oxide (ferumoxide) particles allows labeled cells to be detected by magnetic resonance imaging (MRI) and is commonly used to track stem cell engraftment. However, the validity of MRI for distinguishing surviving ferumoxide-labeled cells from other sources of MRI signal, for example, macrophages containing ferumoxides released from nonsurviving cells, has not been thoroughly investigated. We sought to determine the relationship between the persistence of iron-dependent MRI signals and cell survival 3 weeks after injection of syngeneic or xenogeneic ferumoxides-labeled stem cells (cardiac-derived stem cells) in rats. METHODS AND RESULTS: We studied nonimmunoprivileged human and rat cardiac-derived stem cells and human mesenchymal stem cells doubly labeled with ferumoxides and beta-galactosidase and injected intramyocardially into immunocompetent Wistar-Kyoto rats. Animals were imaged at 2 days and 3 weeks after stem cell injection in a clinical 3-T MRI scanner. At 2 days, injection sites of xenogeneic and syngeneic cells (cardiac-derived stem cells and mesenchymal stem cells) were identified by MRI as large intramyocardial signal voids that persisted at 3 weeks (50% to 90% of initial signal). Histology (at 3 weeks) revealed the presence of iron-containing macrophages at the injection site, identified by CD68 staining, but very few or no beta-galactosidase-positive stem cells in the animals transplanted with syngeneic or xenogeneic cells, respectively. CONCLUSIONS: The persistence of significant iron-dependent MRI signal derived from ferumoxide-containing macrophages despite few or no viable stem cells 3 weeks after transplantation indicates that MRI of ferumoxide-labeled cells does not reliably report long-term stem cell engraftment in the heart.

Therapeutic Properties of Superoxide Dismutase 3 and Mesenchymal Stromal Cells in Peripheral Ischemia

The aim of this study was to characterize the cellular mechanisms leading to the beneficial effect of anti-oxidative gene therapy and pro-angiogenic stem cell therapy in acute peripheral ischemia. Post-ischemic events aim to re-establish tissue blood perfusion, to clear cellular debris, and to regenerate lost tissue by differentiation of satellite cells into myoblasts. Although leukocytes have an essential role in clearing cellular debris and promoting angiogenesis, they also contribute to tissue injury through excessive ROS production. First, we investigated the therapeutic properties of extracellular superoxide dismutase (SOD3) gene transfer. SOD3 was shown to reduce oxidative stress, to normalize glucose metabolism, and to enhance cell proliferation in the ischemic muscle. Analysis of the mitogenic Ras-Erk1/2 pathway showed SOD3 mediated induction offering a plausible explanation for enhanced cell proliferation. In addition, SOD3 reduced NF-κB activity by enhancing IκBα expression thus leading to reduced expression of inflammatory cytokines and adhesion molecules with consequent reduction in macrophage infiltration. Secondly, we sought to determine the fate and the effect of locally transplanted mesenchymal stem/stromal cells (MSCs) in acute ischemia. We showed that a vast majority of the transplanted cells are cleared from the injury site within 24 hours after local transplantation. Despite rapid clearance, transplantation was able to temporarily promote angiogenesis and cell proliferation in the muscle. Lack of graft-derived growth factor expression suggests other than secretory function to mediate this observed effect. In conclusion, both SOD3 and MSCs could be utilized to alleviate peripheral ischemia induced tissue injury. We have described a previously unidentified growth regulatory role for SOD3, and suggest a novel mechanism whereby transplanted MSCs enhance the reparative potential of the recipient tissue through physical contacts.

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

The objective of the study was to isolate, cultivate and characterize equine peripheral blood-derived multipotent mesenchymal stromal cells (PbMSCs). Peripheral blood was collected, followed by the isolation of mononuclear cells using density gradient reagents, and the cultivation of adherent cells. Monoclonal mouse anti-horse CD13, mouse anti-horse CD44, and mouse anti-rat CD90 antibodies were used for the immunophenotypic characterization of the surface of the PbMSCs. These cells were also cultured in specific media for adipogenic and chondrogenic differentiation. There was no expression of the CD13 marker, but CD44 and CD90 were expressed in all of the passages tested. After 14 days of cell differentiation into adipocytes, lipid droplets were observed upon Oil Red O (ORO) staining. Twenty-one days after chondrogenic differentiation, the cells were stained with Alcian Blue. Although the technique for the isolation of these cells requires improvement, the present study demonstrates the partial characterization of PbMSCs, classifying them as a promising type of progenitor cells for use in equine cell therapy.

A novel alternative to cryopreservation for the short-term storage of stem cells for use in cell therapy using alginate encapsulation

Efficient transport of stem/progenitor cells without affecting their survival and function is a key factor in any practical cell-based therapy. However, the current approach using liquid nitrogen for the transfer of stem cells requires a short delivery time window is technically challenging and financially expensive. The present study aims to use semipermeable alginate hydrogels (crosslinked by strontium) to encapsulate, store, and release stem cells, to replace the conventional cryopreservation method for the transport of therapeutic cells within world-wide distribution time frame. Human mesenchymal stem cell (hMSC) and mouse embryonic stem cells (mESCs) were successfully stored inside alginate hydrogels for 5 days under ambient conditions in an air-tight environment (sealed cryovial). Cell viability, of the cells extracted from alginate gel, gave 74% (mESC) and 80% (hMSC) survival rates, which compared favorably to cryopreservation. More importantly, the subsequent proliferation rate and detection of common stem cell markers (both in mRNA and protein level) from hMSCs and mESCs retrieved from alginate hydrogels were also comparable to (if not better than) results gained following cryopreservation. In conclusion, this new and simple application of alginate hydrogel encapsulation may offer a cheap and robust alternative to cryopreservation for the transport and storage of stem cells for both clinical and research purposes.

Origin and regenerative potential of vertebrate mechanoreceptor-associated stem cells

Meissner corpuscles and Merkel cell neurite complexes are highly specialized mechanoreceptors present in the hairy and glabrous skin, as well as in different types of mucosa. Several reports suggest that after injury, such as after nerve crush, freeze injury, or dissection of the nerve, they are able to regenerate, particularly including reinnervation and repopulation of the mechanoreceptors by Schwann cells. However, little is known about mammalian cells responsible for these regenerative processes. Here we review cellular origin of this plasticity in the light of newly described adult neural crest-derived stem cell populations. We also discuss further potential multipotent stem cell populations with the ability to regenerate disrupted innervation and to functionally recover the mechanoreceptors. These capabilities are discussed as in context to cellularly reprogrammed Schwann cells and tissue resident adult mesenchymal stem cells.

Alternative generation of CNS neural stem cells and PNS derivatives from neural crest-derived peripheral stem cells

Neural crest-derived stem cells (NCSCs) from the embryonic peripheral nervous system (PNS) can be reprogrammed in neurosphere (NS) culture to rNCSCs that produce central nervous system (CNS) progeny, including myelinating oligodendrocytes. Using global gene expression analysis we now demonstrate that rNCSCs completely lose their previous PNS characteristics and acquire the identity of neural stem cells derived from embryonic spinal cord. Reprogramming proceeds rapidly and results in a homogenous population of Olig2-, Sox3-, and Lex-positive CNS stem cells. Low-level expression of pluripotency inducing genes Oct4, Nanog, and Klf4 argues against a transient pluripotent state during reprogramming. The acquisition of CNS properties is prevented in the presence of BMP4 (BMP NCSCs) as shown by marker gene expression and the potential to produce PNS neurons and glia. In addition, genes characteristic for mesenchymal and perivascular progenitors are expressed, which suggests that BMP NCSCs are directed toward a pericyte progenitor/mesenchymal stem cell (MSC) fate. Adult NCSCs from mouse palate, an easily accessible source of adult NCSCs, display strikingly similar properties. They do not generate cells with CNS characteristics but lose the neural crest markers Sox10 and p75 and produce MSC-like cells. These findings show that embryonic NCSCs acquire a full CNS identity in NS culture. In contrast, MSC-like cells are generated from BMP NCSCs and pNCSCs, which reveals that postmigratory NCSCs are a source for MSC-like cells up to the adult stage.

Controversial role of toll-like receptor 4 in adult stem cells

Adult or somatic stem cells are tissue-resident cells with the ability to proliferate, exhibit self-maintenance as well as to generate new cells with the principal phenotypes of the tissue in response to injury or disease. Due to their easy accessibility and their potential use in regenerative medicine, adult stem cells raise the hope for future personalisable therapies. After infection or during injury, they are exposed to broad range of pathogen or damage-associated molecules leading to changes in their proliferation, migration and differentiation. The sensing of such damage and infection signals is mostly achieved by Toll-Like Receptors (TLRs) with Toll-like receptor 4 being responsible for recognition of bacterial lipopolysaccharides (LPS) and endogenous danger-associated molecular patterns (DAMPs). In this review, we examine the current state of knowledge on the TLR4-mediated signalling in different adult stem cell populations. Specifically, we elaborate on the role of TLR4 and its ligands on proliferation, differentiation and migration of mesenchymal stem cells, hematopoietic stem cells as well as neural stem cells. Finally, we discuss conceptual and technical pitfalls in investigation of TLR4 signalling in stem cells.

Systemic delivery of adult stem cells improves cardiac function in spontaneously hypertensive rats

Heart regeneration after myocardial infarction (MI) can occur after cell therapy, but the mechanisms, cell types and delivery methods responsible for this improvement are still under investigation. In the present study, we evaluated the impact of systemic delivery of bone marrow cells (BMC) and cultivated mesenchymal stem cells (MSC) on cardiac morphology, function and mortality in spontaneously hypertensive rats (SHR) submitted to coronary occlusion. Female syngeneic adult SHR, submitted or not (control group; C) to MI, were treated with intravenous injection of MSC (MI + MSC) or BMC (MI + BM) from male rats and evaluated after 1, 15 and 30 days by echocardiography. Systolic blood pressure (SBP), functional capacity, histology, mortality rate and polymerase chain reaction for the Y chromosome were also analysed. Myocardial infarction induced a decrease in SBP and BMC, but not MSC, prevented this decrease. An improvement in functional capacity and ejection fraction (38 +/- 4, 39 +/- 3 and 58 +/- 2% for MI, MI + MSC and MI + BM, respectively; P < 0.05), as well as a reduction of the left ventricle infarcted area, were observed in rats from the MI + BM group compared with the other three groups. Treated animals had a significantly reduced lesion tissue score. The mortality rate in the C, MI + BM, MI + MSC and MI groups was 0, 0, 16.7 and 44.4%, respectively (P < 0.05 for the MI + MSC and MI groups compared with the C and MI + BM groups). The results of the present study suggest that systemic administration of BMC can improve left ventricular function, functional capacity and, consequently, reduce mortality in an animal model of MI associated with hypertension. We speculate that the cells transiently home to the myocardium, releasing paracrine factors that recruit host cells to repair the lesion.

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.

Isolation and characterization of Wharton's jelly-derived multipotent mesenchymal stromal cells obtained from bovine umbilical cord and maintained in a defined serum-free three-dimensional system

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

In vitro analysis with human bone marrow stem cells on Ti-15Mo alloy for dental and orthopedic implants application

Aim: Nowadays, research on orthopedic and dental implants is focused on titanium alloys for their mechanical properties and corrosion resistance in the human body environment. Another important aspect to be investigated is their surface topography, which is very important to osseointegration. With laser beam irradiation for roughening the implants surface an easier control of the microtopography is achieved, and surface contamination is avoided. The aim of this study was to assess human bone marrow stem cells response to a newly developed titanium alloy, Ti-15Mo, with surface topography modified by laser beam irradiation. Materials and methods: A total of 10 Ti machined disks (control), 10 Ti-15Mo machined disks and 10 Ti-15Mo disks treated by laser beam-irradiation were prepared. To study how Ti-15Mo surface topografy can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells marker were analyzed, using real time Reverse Transcription-Polymerase Chain Reaction. Results: In Test 1 (comparison between Ti-15Mo machined disks and Ti-machined disks) quantitative real-time RT-PCR showed a significant induction of ALPL, FOSL1 and SPP1, which increase 20% or more. In Test 2 (comparison between Ti-15Mo laser treated disks and Ti-machined disks) all investigated genes were up-regulated. By comparing Test 1 and Test 2 it was detected that COL1A1, COL3A1, FOSL1 and ENG sensibly increased their expression whereas RUNX2, ALPL and SPP1 expression remained substantially unchanged. Conclusion: The present study demonstrated that laser treated Ti-15Mo alloys are promising materials for implants application.

Isolation and characterization of equine peripheral blood-derived multipotent mesenchymal stromal cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)