Reliable Cell Source Research Articles

Transplantation of neural stem cells derived from human cord blood to the brain of adult and neonatal rats.

Searching for a reliable source of alternative neural stem cells for experimental treatment of neurological disorders we have established neural stem cell line derived from human umbilical cord blood (HUCB-NSC) (Buzanska et al. 2006). These cells have been shown to differentiate along neuronal and glial lineages in the promoting in vitro conditions. In the current study we transplanted HUCB-NSC into rat brain to determine whether the neural progenitors would be able to survive, migrate and eventually adopt neural phenotypes after exposure to central nervous system (CNS) microenvironment. Our experiments revealed that HUCB-NSC grafting into the brain of adult rats limited their survival up-to two weeks probably due to their elimination by severe immunological host reaction evoked by xenotransplantation. HUCB-NSC graft in neonates survived longer time in rat brain, migrated, proliferated and differentiated into neuronal cells however their presence in the host tissue did not exceed more than five weeks after transplantation.

Potential sources of stem cells as a regenerative therapy for Parkinson's disease.

Stem cells are believed to hold enormous promise as potential replacement therapy in the treatment of neurodegenerative diseases such as Parkinson’s disease (PD). Stem cells were investigated to be the alternative therapeutic source capable of differentiating into dopamine (DA) neurons. Multiple important signaling factors were recorded for the induction of DA neuronal traits from mouse embryonic stem cells (ESCs) such as fibroblast growth factor 8, sonic hedgehog, and Wnt 1. Recent protocols were described for the differentiation of human ESCs into DA neurons, achieving high efficiency of DA neuronal derivation. Despite that, the use of human ESCs is still ethically controversial. The transcription factors necessary for DA neuron development from adult neural stem cells (NSCs), such as Pitx3, Nurr1, En-1, En-2, Lmx1a, Lmx1b, Msx1, and Ngn2, were investigated. In addition to replacement of lost DA neurons, adult NSCs were recorded to provide neuroprotective and neurogenic factors for the mesencephalon. In addition, induced pluripotent stem cells and bone marrow-derived mesenchymal stem cells represent reliable stem cell sources of DA neurons. Future studies are recommended to provide further insight into the regenerative capacity of stem cells needed for the treatment of PD.

Long-Term Survival of Human Neural Stem Cells in the Ischemic Rat Brain upon Transient Immunosuppression

Understanding the physiology of human neural stem cells (hNSCs) in the context of cell therapy for neurodegenerative disorders is of paramount importance, yet large-scale studies are hampered by the slow-expansion rate of these cells. To overcome this issue, we previously established immortal, non-transformed, telencephalic-diencephalic hNSCs (IhNSCs) from the fetal brain. Here, we investigated the fate of these IhNSC's immediate progeny (i.e. neural progenitors; IhNSC-Ps) upon unilateral implantation into the corpus callosum or the hippocampal fissure of adult rat brain, 3 days after global ischemic injury. One month after grafting, approximately one fifth of the IhNSC-Ps had survived and migrated through the corpus callosum, into the cortex or throughout the dentate gyrus of the hippocampus. By the fourth month, they had reached the ipsilateral subventricular zone, CA1-3 hippocampal layers and the controlateral hemisphere. Notably, these results could be accomplished using transient immunosuppression, i.e administering cyclosporine for 15 days following the ischemic event. Furthermore, a concomitant reduction of reactive microglia (Iba1+ cells) and of glial, GFAP+ cells was also observed in the ipsilateral hemisphere as compared to the controlateral one. IhNSC-Ps were not tumorigenic and, upon in vivo engraftment, underwent differentiation into GFAP+ astrocytes, and β-tubulinIII+ or MAP2+ neurons, which displayed GABAergic and GLUTAmatergic markers. Electron microscopy analysis pointed to the formation of mature synaptic contacts between host and donor-derived neurons, showing the full maturation of the IhNSC-P-derived neurons and their likely functional integration into the host tissue. Thus, IhNSC-Ps possess long-term survival and engraftment capacity upon transplantation into the globally injured ischemic brain, into which they can integrate and mature into neurons, even under mild, transient immunosuppressive conditions. Most notably, transplanted IhNSC-P can significantly dampen the inflammatory response in the lesioned host brain. This work further supports hNSCs as a reliable and safe source of cells for transplantation therapy in neurodegenerative disorders.

Validation of human periodontal ligament‐derived cells as a reliable source for cytotherapeutic use

Periodontal ligament (PDL) is a reliable cell source for periodontal regeneration. In this study, an optimal protocol for the extraction, expansion, and characterization of human PDL (hPDL) cells was examined for clinical trials. hPDL tissues were obtained from 41 surgically extracted teeth and digested with enzymes. Human adipose-derived stem cells (hADSCs), bone marrow-derived mesenchymal stem cells (hBMMSCs), and gingival fibroblasts (hGFs) were used for comparison. For each sample, the proliferative capacity, colony-forming ability, alkaline phosphatase activity, differentiation ability, the cell surface antigens, gene expression, and regenerative potential were examined. hPDL cells were more successfully extracted with collagenase/dispase [29/30 (96.7%)] than with trypsin/EDTA [8/11 (72.7%)], and exhibited osteogenic potential both in vitro and in vivo. The proliferation of hPDL cells was rapid at a low cell density. hPDL cells frequently differentiated into cementoblastic/osteoblastic lineage (∼60%). In contrast, their adipogenic and chondrogenic potentials were lower than those of hADSCs and hBMMSCs. Some genes (NCAM1, S100A4, and periostin) were preferentially expressed in hPDL cells compared with those of hBMMSCs and hGFs. Immunohistochemical studies revealed the expressions of S100A4 and periostin in hPDL tissue. A protocol for the successful cultivation and validation of hPDL cells is proposed for clinical settings.

Differentiation of Adipose-Derived Stem Cells into Contractile Smooth Muscle Cells Induced by Transforming Growth Factor-β1 and Bone Morphogenetic Protein-4

Smooth muscle cells (SMCs) play an essential role in maintaining the structural and functional integrity of blood vessel and thus is a critical element for blood vessel construction via tissue engineering approach. Adipose-derived stem cells (ASCs) represent a reliable source of mesenchymal stem cells with multidifferentiation potential. In this study, the feasibility of differentiation of human ASCs (hASCs) into cells with phenotypic and functional properties of SMCs was explored. hASCs isolated from human lipoaspirate were expanded to passage 5 and then induced with administration of transforming growth factor-beta1 (TGF-beta1) and bone morphogenetic protein-4 (BMP4) either alone or in combination with culture medium. Expression of SMC-related markers including alpha-SM actin (alpha-SMA, SM22alpha, calponin, and SM myosin heavy chain) were detected by immunofluorescent staining, reverse transcription (RT)-polymerase chain reaction, and western blot analysis. It was found that only under the circumstance of a combined stimulation with TGF-beta1 and BMP4, both early and mid markers (alpha-SMA, SM22alpha, calponin) as well as a late marker (SM myosin heavy chain) of SMC differentiation were identified to similar levels as those in human umbilical artery SMCs. More importantly, these SM differentiated cells showed the function of contracting collagen matrix lattice when they were entrapped inside. The contractile function of differentiated hASCs was further enhanced by direct exposure to 60 mM KCl, consistent with what occurred in human umbilical artery SMCs. These results provide evidence that ASCs possess the potential to differentiate into contractile SM-like cells when stimulated by TGF-beta1 and BMP4 together. SMCs differentiated from hASCs may provide an abundant source as seed cells for blood vessel engineering.

Derivation of Engraftable Myogenic Precursors from Murine ES/iPS cells and Generation of Disease-specific iPS cells from Patients with Duchenne Muscular dystrophy (DMD) and Other Diseases

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a progressive, lethal muscle disorder with no effective cure despite extensive research efforts in the field. In recent years, many different myogenic cells originating from adult tissues have been reported. However, the establishment of a reliable cell source is required for clinical application. Embryonic stem (ES) cells and the recently established induced pluripotent stem (iPS) cells are totipotent stem cells that are infinitely expandable and capable of differentiating into all types of somatic cells. In this study, we established a novel protocol to derive myogenic precursors from murine ES iPS cells with a monoclonal antibody SM C-2.6 that recognizes quiescent satellite cells. SM C-2.6-positive cells are highly myogenic and efficiently differentiate into myofibers both in vitro and in vivo. Furthermore, the transplanted cells demonstrated extensive muscle regeneration activity in a second injury model without cell transplantation as well as long-term engraftment up to 24 weeks. Both these results indicated that the transplanted cells act as muscle stem cells as well as myogenic precursors. Our data suggest that iPS cells are a new attractive cell source for cell-based therapies. Recently, we succeeded the generation of disease-specific iPS cells from skin fibroblasts of a DMD patient and his parents by the reprogramming with Oct3 4, Sox2, Klf4 and cMyc. Generation of patient-specific iPS cells indicates possible strategy of regenerative medicine for DMD with normal or patient iPS cells. Finally, I will talk about other diseasespecific iPS cells from the patients with CINCA (Chronic Infantile Neurological Cutaneous and Articular) syndrome with somatic mosaicism

Adipose stem cell side population in the mouse

Adipose tissue has become a reliable source of adult stem cells, which appear to possess a yet-undetermined degree of plasticity. With the difficulties associated with harvesting adult bone marrow stem cells, adipose tissue may represent a valuable and easily acquired source of stem cells. Stem cells have been identified using the DNA binding dye Hoechst 33342 and flow cytometry in various tissues known as the side population (SP). The present study shows, for the first time, the presence of side population stem cells in adult adipose tissues. Flow cytometric identification and isolation of this subpopulation of stem cells revealed that in the mouse there are 2.5% of adipose SP cells within the stromal vascular fraction of adipose tissue. In culture, mouse adipose SP cells showed the capacity to undergo in vitro differentiation into osteogenic, chondrogenic and adipogenic lineages. In NOD/SCID mice, freshly sorted mouse adipose SP cells were able to engraft and assist in wound healing. This animal model study showed that adipose SP cells were able to regenerate epithelial layers and connective tissue with minor scar formation. The ability of this novel cell population within adipose tissue to undergo directional differentiation in vitro and to regenerate skin in vivo has potential impact for uses in surgical dermal applications.

Transfection of adult canine Schwann cells and olfactory ensheathing cells at early and late passage with human TERT differentially affects growth factor responsiveness and in vitro growth

Adult canine Schwann cells and olfactory ensheathing cells (OECs) are closely related cell types that are considered attractive candidates for translational studies of neural repair. To establish a reliable cell source by comparing the in vitro properties of immortalized Schwann cells and OECs for transplantation purposes, we transfected both cell types with human telomerase reverse transcriptase (hTERT). Ectopic hTERT expression has been shown to induce immortalization of various cell types without substantial alterations of their phenotypes. Schwann cells and OECs were isolated from adult dogs, transfected with hTERT at early (P4) and late passage (P26), characterized regarding in vitro proliferation, antigenic expression and senescence-associated genes in the presence and absence of fibroblast growth factor-2 (FGF-2). Ectopic hTERT expression in late passage glia treated with but not without FGF-2 prevented the decline in proliferation observed in non-transfected cells. Immortalization did not alter p75 NTR and GFAP but O4 and A2B5 expression. Contrary to this, early passage hTERT transfection significantly reduced proliferation independent of FGF-2 and lowered expression of O4 and GFAP in both cell types. Transfection did not alter mRNA expression of senescence-associated genes such as p53 and p16. No substantial differences were found between Schwann cells and OECs underscoring the close relationship of both cell types. Taken together, we established a stable source of adult canine Schwann cells and OECs and demonstrated that the effects of hTERT expression on in vitro growth and growth factor responsiveness depend on the replicative age.

Myocyte Enhancer Factor 2C as a Neurogenic and Antiapoptotic Transcription Factor in Murine Embryonic Stem Cells

Cell-based therapies require a reliable source of cells that can be easily grown, undergo directed differentiation, and remain viable after transplantation. Here, we generated stably transformed murine ES (embryonic stem) cells that express a constitutively active form of myocyte enhancer factor 2C (MEF2CA). MEF2C has been implicated as a calcium-dependent transcription factor that enhances survival and affects synapse formation of neurons as well as differentiation of cardiomyocytes. We now report that expression of MEF2CA, both in vitro and in vivo, under regulation of the nestin enhancer effectively produces "neuronal" progenitor cells that differentiate into a virtually pure population of neurons. Histological, electrophysiological, and behavioral analyses demonstrate that MEF2C-directed neuronal progenitor cells transplanted into a mouse model of cerebral ischemia can successfully differentiate into functioning neurons and ameliorate stroke-induced behavioral deficits.

Vascular smooth muscle cells for use in vascular tissue engineering obtained by endothelial-to-mesenchymal transdifferentiation (EnMT) on collagen matrices

The discovery of the endothelial progenitor cell (EPC) has led to an intensive research effort into progenitor cell-based tissue engineering of (small-diameter) blood vessels. Herein, EPC are differentiated to vascular endothelial cells and serve as the inner lining of bioartificial vessels. As yet, a reliable source of vascular smooth muscle progenitor cells has not been identified. Currently, smooth muscle cells (SMC) are obtained from vascular tissue biopsies and introduce new vascular pathologies to the patient. However, since SMC are mesenchymal cells, endothelial-to-mesenchymal transdifferentiation (EnMT) may be a novel source of SMC. Here we describe the differentiation of smooth muscle-like cells through EnMT. Human umbilical cord endothelial cells (HUVEC) were cultured either under conditions favoring endothelial cell growth or under conditions favoring mesenchymal differentiation (TGF-β and PDGF-BB). Expression of smooth muscle protein 22α and α-smooth muscle actin was induced in HUVEC cultured in mesenchymal differentiation media, whereas hardly any expression of these markers was found on genuine HUVEC. Transdifferentiated endothelial cells lost the ability to prevent thrombin formation in an in vitro coagulation assay, had increased migratory capacity towards PDGF-BB and gained contractile behavior similar to genuine vascular smooth muscle cells. Furthermore, we showed that EnMT could be induced in three-dimensional (3D) collagen sponges. In conclusion, we show that HUVEC can efficiently transdifferentiate into smooth muscle-like cells through endothelial-to-mesenchymal transdifferentiation. Therefore, EnMT might be used in future progenitor cell-based vascular tissue engineering approaches to obtain vascular smooth muscle cells, and circumvent a number of limitations encountered in current vascular tissue engineering strategies.

Adipose-derived stem cells: Isolation, expansion and differentiation

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue has proven to serve as an abundant, accessible and rich source of adult stem cells with multipotent properties suitable for tissue engineering and regenerative medical applications. There has been increased interest in adipose-derived stem cells (ASCs) for tissue engineering applications. Here, methods for the isolation, expansion and differentiation of ASCs are presented and described in detail. While this article has focused on the isolation of ASCs from human adipose tissue, the procedure can be applied to adipose tissues from other species with minimal modifications.

Efficient Delivery of Human Single Fiber-Derived Muscle Precursor Cells via Biocompatible Scaffold

The success of cell therapy for skeletal muscle disorders depends upon two main factors: the cell source and the method of delivery. In this work we have explored the therapeutic potential of human muscle precursor cells (hMPCs), obtained from single human muscle fibers, implanted in vivo via micropatterned scaffolds. hMPCs were initially expanded and characterized in vitro by immunostaining and flow cytometric analysis. For in vivo studies, hMPCs were seeded onto micropatterned poly-lactic-glycolic acid 3D-scaffolds fabricated using soft-lithography and thermal membrane lamination. Seeded scaffolds were then implanted in predamaged tibialis anterior muscles of CD1 nude mice; hMPCs were also directly injected in contralateral limbs as controls. Similarly to what we previously described with mouse precursors cells, we found that hMPCs were able to participate in muscle regeneration and scaffold-implanted muscles contained a greater number of human nuclei, as revealed by immunostaining and Western blot analyses. These results indicate that hMPCs derived from single fibers could be a good and reliable cell source for the design of therapeutic protocols and that implantation of cellularized scaffolds is superior to direct injection for the delivery of myogenic cells into regenerating skeletal muscle.

Mesothelial Cells from Tunica Vaginalis, a Practical Source for Mesothelial Transplantation

Transplantation of mesothelial cells is used to repair peritoneum that is damaged by surgery, peritonitis, and peritoneal dialysis. The largest obstacle for clinical application of mesothelial cell transplantation is the lack of a reliable source of mesothelial cells. So far, they are isolated from omentum, mesentery, parietal wall and ascites. Procedures used to obtain mesothelial cells from the omentum or mesentery are invasive, however, especially in pre-operative situations. Sufficient amounts of ascites for aspiration can not be obtained under physiological conditions. We have developed a novel method of isolating mesothelial cells from the tunica vaginalis. The tunica vaginalis originates from the peritoneum and descends into the scrotum along with the testis during fetal development. This region provides a source of mesothelial cells that is convenient to approach and free from abdominal complications. Transplantation of autologous mesothelial cells that were isolated from tunica vaginalis was effective in preventing post-operative adhesions. In this review, we summarize mesothelial cell transplantation trials and describe the method of isolating mesothelial cells form the tunica vaginalis. Mesothelial cell transplantation might be widely accepted for clinical use in the near future.

Adipose-Derived Stem Cells for Regenerative Medicine

The emerging field of regenerative medicine will require a reliable source of stem cells in addition to biomaterial scaffolds and cytokine growth factors. Adipose tissue represents an abundant and accessible source of adult stem cells with the ability to differentiate along multiple lineage pathways. The isolation, characterization, and preclinical and clinical application of adipose-derived stem cells (ASCs) are reviewed in this article.

O.273 Bone tissue engineering from mesenchymal stem cells derived from human pulp tissue

Functional tissue engineering of connective tissues such as the anterior cruciate ligament (ACL) remains a significant clinical challenge, largely due to the need for mechanically competent scaffold systems for grafting, as well as a reliable cell source for tissue formation. We have designed an aligned, polylactide-co-glycolide (PLGA) nanofiber-based scaffold with physiologically relevant mechanical properties for ligament regeneration. The objective of this study is to identify optimal tissue engineering strategies for fibroblastic induction of human mesenchymal stem cells (hMSC), testing the hypothesis that basic fibroblast growth factor (bFGF) priming coupled with tensile loading will enhance hMSC-mediated ligament regeneration. It was observed that compared to the unloaded, as well as growth factor-primed but unloaded controls, bFGF stimulation followed by physiologically relevant tensile loading enhanced hMSC proliferation, collagen production and subsequent differentiation into ligament fibroblast-like cells, upregulating the expression of types I and III collagen, as well as tenasin-C and tenomodulin. The results of this study suggest that bFGF priming increases cell proliferation, while mechanical stimulation of the hMSCs on the aligned nanofiber scaffold promotes fibroblastic induction of these cells. In addition to demonstrating the potential of nanofiber scaffolds for hMSC-mediated functional ligament tissue engineering, this study yields new insights into the interactive effects of chemical and mechanical stimuli on stem cell differentiation.

Clonogenic multipotent stem cells in human adipose tissue differentiate into functional smooth muscle cells.

Smooth muscle is a major component of human tissues and is essential for the normal function of a multitude of organs including the intestine, urinary tract and the vascular system. The use of stem cells for cell-based tissue engineering and regeneration strategies represents a promising alternative for smooth muscle repair. For such strategies to succeed, a reliable source of smooth muscle precursor cells must be identified. Adipose tissue provides an abundant source of multipotent cells. In this study, the capacity of processed lipoaspirate (PLA) and adipose-derived stem cells to differentiate into phenotypic and functional smooth muscle cells was evaluated. To induce differentiation, PLA cells were cultured in smooth muscle differentiation medium. Smooth muscle differentiation of PLA cells induced genetic expression of all smooth muscle markers and further confirmed by increased protein expression of smooth muscle cell-specific alpha actin (ASMA), calponin, caldesmon, SM22, myosin heavy chain (MHC), and smoothelin. Clonal studies of adipose derived multipotent cells demonstrated differentiation of these cells into smooth muscle cells in addition to trilineage differentiation capacity. Importantly, smooth muscle-differentiated cells, but not their precursors, exhibit the functional ability to contract and relax in direct response to pharmacologic agents. In conclusion, adipose-derived cells have the potential to differentiate into functional smooth muscle cells and, thus, adipose tissue can be a useful source of cells for treatment of injured tissues where smooth muscle plays an important role.

How can we get more beta cells?

The need for a reliable source of functional beta cells has led to many new investigations in an effort to drive the differentiation of embryonic stem cells, of putative stem cells, or of pancreatic progenitor cells to form new beta cells. There appears to be a plasticity of pancreatic cells in vitro that may be exploited to generate the necessary beta cells. Major questions still remain: whether there are true pancreatic stem cells, what are the pancreatic progenitor cells after birth, and whether expanded beta cells themselves could serve as the source.

Human Hepatocyte Transplantation for Acute Liver Failure: State of the Art and Analysis of Cell Sources

Liver transplantation is the only treatment available for acute liver failure. However, mortality rates remain high because of the shortage of donor organs. Indeed up to 20% of patients with acute liver failure may survive without transplantation. In the last two decades, research has focused on the development of alternative or supportive measures to deal with acute liver failure; one of the most studied is hepatocyte transplantation, because it is thought that the function of the liver can only be replaced with a biological substrate characterized by functioning liver cells. Hepatocyte transplantation has been successful in many animal models of acute liver failure, although only several clinical attempts have been made in humans with encouraging but not yet convincing results, mainly because of the lack of a reliable source of live liver cells. Allogenic and xenogenic fresh or cryopreserved hepatocytes have been tested. Recent research has focused on fetal hepatocytes and progenitor liver cells of both hepatic and bone marrow origin. The ability to preserve and bank human hepatocytes would allow pooling of cells from multiple donors to increase the numbers for transplantation. The development of a reliable and large-scale available source of live liver cells would probably have a major impact on the introduction of hepatocyte transplantation in clinical practice.

A modified method for generation of neural precursor cells from cultured mouse embryonic stem cells

The pluripotency and high proliferative capacity of embryonic stem (ES) cells make them an attractive source of different cell types for biomedical research and cell replacement therapies. It has been demonstrated that ES cells can be induced into neural precursor cells (NPCs) under conditions. NPCs can be expanded in large numbers for significant periods of time to provide a reliable source of cells for transplantation in neurodegenerative disorders and injury of the central nervous system. This study describes a modified method for generation of NPCs from cultured mouse ES cells.

Neural precursor cells differentiated from mouse embryonic stem cells relieve symptomatic motor behavior in a rat model of Parkinson’s disease

Pluripotent embryonic stem (ES) cells are the most versatile cells, with the potential to differentiate into all types of cell lineages including neural precursor cells (NPCs), which can be expanded in large numbers for significant periods of time to provide a reliable cell source for transplantation in neurodegenerative disorders such as Parkinson’s disease (PD). In the present study, we used the MESPU35 mouse ES cell line, which expresses enhanced green fluorescent protein that enables one to distinguish between transplanted cells and cells of host origin. Embryoid bodies (EBs) were formed and were induced to NPCs in N2 selection medium plus fibronectin. Praxiology and immunohistochemistry methods were used to observe the survival, differentiation, and therapeutic effect of NPCs after grafted into the striatum of PD rats. We found that mouse ESc were differentiated into nestin-positive NPCs 6 days after the EBs formed and cultured in the N2 selection medium. The number of survival NPCs was increased significantly by fibronectin. About 23.76 ± 2.29% of remaining cells were tyrosine hydroxylase (TH)-positive 12 days after NPCs were cultured in N2 selective medium. The survival rates of NPCs were 2.10 ± 0.41% and about 90.90 ± 3.00% of the engrafted NPCs were TH-positive 6 weeks after transplantation into the striatum of PD rats. The rotation of PD rats was relieved 3 weeks after the NPCs transplantation and this effect was kept for at least 6 weeks. It suggests that most of the survival NPCs derived from ES cells differentiated into TH-positive neurons after grafted into the striatum of PD rats, which produces therapeutic effect on PD.