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NOTE: An enormous literature is rapidly developing on the topic of adult stem cells and their potential for developmental plasticity and regenerative medicine. This bibliography lists recent comprehensive reviews and some representative articles.
- Stretching the limits: stem cells in regeneration science. Stocum DL, Zupanc GK. Dev Dyn. 2008;237:3648-3671. Review.
This is an extensive review (23 pages) of the status of research in Regenerative Medicine. In the past year alone, a torrent of basic and clinical research papers has flooded the literature. The following excerpts give a flavor or the article, but cannot begin to provide the detail.
The focus of regenerative medicine is rebuilding damaged tissues by cell transplantation or implantation of bioartificial tissues. In either case, therapies focus on adult stem cells (ASCs) and embryonic stem cells (ESCs) as cell sources.
Adult Stem Cells (ASCs): Regeneration of central nervous tissue. The neurogenic potential of neural stem cells (NSCs) could form the basis for a replacement therapy for neurons lost to injury or neurodegenerative disease. Intense effort has been put into developing NSC-based therapies for spinal cord injury and neurodegenerative disease. Allogeneic NSCs transplanted to spinal cord lesions have been reported to promote partial recovery from paralysis. However, the modest improvements reported are likely not due to the differentiation of donor NSCs into new neurons, but to effects on host cells. Rats injected intravenously with umbilical cord blood 5 days after lesioning the spinal cord were reported to achieve partial recovery of locomotory behavior. However, histological examination indicated that the labeled cells, of which fewer than 1,000 survived, did not differentiate into either neurons or glia.
Regeneration of myocardium. Recent reviews have summarized the effects of clinical trials in which satellite cells (the stem cell of skeletal muscle) and bone marrow cells were transplanted to ameliorate the effects of myocardial infarction. The results have been generally disappointing, with improvements in cardiac function ranging from nonexistent to modest. Neither satellite cells nor bone marrow cells differentiated into new cardiomyocytes. To test the idea of cardiomyocyte renewal from stem cells, differentiated mouse cardiomyocytes were marked with an inducible transgene for green fluorescent protein (GFP). After myocardial infarction and induction of the GFP reporter with tamoxifen, approximately 15% of the GFP-labeled cardiomyocytes were replaced by unlabeled cardiomyocytes. These results suggest that new cardiomyocytes can indeed be produced from cardiac stem cells.
Regeneration of musculoskeletal tissues. Allogeneic cellular transplants to regenerate normal muscle in place of dystrophic muscle in mdx mice and muscular dystrophy patients have not been successful due to inflammation, immune rejection, poor survival and limited migration of the transplanted cells. Because mesenchymal stem cells (MSCs) normally give rise to chondroblasts and osteoblasts, they are logical candidates to repair defects in articular cartilage and bone. The results of transplanting MSCs alone or in scaffolds into defects that involve only the hyaline cartilage have been generally disappointing. Infants with osteogenesis imperfecta have been successfully treated by engraftment of normal bone marrow cells, and grafts of autogeneic bone marrow cells have promoted the healing of nonunion fractures. The MSCs of the bone marrow are likely responsible for rebuilding new bone tissue.
Embryonic Stem Cells (ESCs): Basic science studies and preclinical studies utilizing ESCs (which are outside the prevue of the Cord Blood Forum) are reviewed in some detail by the authors. The conclude that ESCs show great promise as a cell source for the regeneration of new tissue, due to their high growth and self-renewal capacity, and their ability to differentiate into a myriad of precursor or differentiated cell types when directed by the appropriate set of environmental factors.
Issues surrounding the use of ASCs and ESCs for cell-based therapy. Several problems currently limit the potential of ASCs for regenerative therapies. It is generally difficult to get ASCs in the numbers required for cell therapy. A better understanding of the composition and three dimensional spatial organization of ASC microniches will be essential to achieving their expansion in vitro without loss of regenerative potential. It would be preferable to have a pluripotent cell source that would be a “universal donor.” ESCs may be contaminated from mouse feeder layers or animal serum-supplemented medium with pathogens or xenogens (cells or proteins) that might trigger host immune reactions. Undifferentiated human ESCs form teratomas when injected subcutaneously or intramuscularly into SCID mice. Directed differentiation protocols for clinical use require 100% purity of differentiated cell populations to avoid the possibility of teratoma formation. ASCs and ESCs face some common hurdles as therapies. One is how to accurately deliver and/or home the cells or their derivatives to the injury site, and another is immunorejection.
Two other topics reviewed are somatic cell nuclear transfer (SCNT) and the generation of iPS cells in vitro. Two very big hurdles to the routine creation of patient-specific ESCs by SCNT are (1) a shortage of human eggs and (2) a low efficiency of reprogramming. Problems facing the use of iPSCs are (1) the necessity to have a 100% differentiation frequency to avoid the possibility of teratocarcinoma formation, (2) different lines of human iPS cells will exhibit highly variable HLA profiles and differentiation bias and (3) there is the important question of how robust is the differentiation of iPS cells, and how they will behave over long periods of time.
The authors provide a perspective on the future including the feasibility of a chemical induction approach to regeneration in vitro and in vivo, and conclude with a short segment on the limits of regenerative medicine.
- Very small embryonic-like (VSEL) stem cells in adult organs and their potential role in rejuvenation of tissues and longevity. Ratajczak MZ, Zuba-Surma EK, Shin DM, Ratajczak J, Kucia M. Exp Gerontol. 2008;43:1009-1017.
This is one of a number of articles during the last few years by this research group on the identification and characterization of very small embryonic-like (VSEL) stem cells and their potential in regenerative medicine.
The authors purified rare CXC chemokine receptor 4 expressing (CXCR4(+)) small stem cells from the murine bone marrow that express markers characteristic for embryonic stem cells, epiblast stem cells, and primordial germ cells. These primitive cells were named “very small embryonic-like “ (VSEL) stem cells.
Data indicate that VSELs are also present in many other organs in mice and that they may differentiate into cells from all three germ layers. Similar stem cells were also isolated from human cord blood and mobilized peripheral blood. The authors suggest that VSELs are deposited during gastrulation and organogenesis in developing organs/tissues of mammals as a population of pluripotent stem cells that give rise to tissue committed monopotent stem cells and that their number decreases with age. Therefore VSELs could play a pivotal role in normal rejuvenation of adult tissues as well as involvement in regeneration of damaged organs.
Thus, these cells are potential stem cell candidates for regenerative medicine. The authors also envision that the regenerative potential of these cells could be harnessed to decelerate the aging processes.
- Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro. McGuckin C, Jurga M, Ali H, Strbad M, Forraz N. Nat Protoc. 2008;3:1046-1055.
The authors describe a 3-week protocol which produces embryonic-like stem cells from human umbilical cord blood (CBEs) for neural differentiation using a three-step system (cell isolation/expansion/differentiation). Much of this article consists of a detailed protocol for the isolation of these cells.
The isolation procedure produces a highly purified fraction (CD45-, CD33-, CD7-, CD235a-) of small pluripotent stem cells (2-3 microm in diameter) coexpressing embryonic stem cell markers including Oct4 and Sox2. Initial CBE expansion is performed in high density (5-10 millions per ml) in the presence of extracellular matrix proteins and epidermal growth factor. Subsequent neural differentiation of the CBEs requires sequential introduction of morphogenes, retinoic acid, brain-derived neurotrophic factor and cyclic AMP. The methods described emphasize defined media and reagents at all stages of the experiment comparable to protocols described for culturing human embryonic stem cells and cells from other somatic stem cell sources.
The authors state that neural progenitor and cells generated from CBEs may be used for in vitro drug testing and cell-based assays and potentially for clinical transplantation.
- Induced pluripotent stem cells: current progress and potential for regenerative medicine. Amabile G, Meissner A. Trends Mol Med. 2009;15:59-68.
Nuclear-transfer experiments over the past 50 years have established that, despite the decrease in developmental potential, the nucleus of most, if not all, adult cells retains nuclear plasticity and can be reset to an embryonic state. In accordance with this, cells (or nuclei) can be converted to a pluripotent state. In the late 1990s, two independent studies raised the possibility of generating human patient-specific stem cells. Although proof-of-principle has been demonstrated in the mouse, human somatic cell-nuclear transfer (SCNT) has yet to be accomplished. Although there seem to be no conceptual obstacles, the procedure is technically challenging, inefficient and dependent on voluntary donation of a large number of unfertilized oocytes.
The landmark development of induced pluripotent stem (iPS) cells (pluripotent cells derived from any differentiated cell type through ectopic expression of transcription factors) has opened a new frontier in the field of regenerative medicine. Mouse and human iPS cells possess morphological, molecular and developmental features that closely resemble those of blastocyst-derived embryonic stem (ES) cells. Although the exact mechanism of reprogramming remains unknown, only a few challenges, including generation of integration-free human iPS cells and improved ways of characterizing them, remain before iPS cells could be used routinely in pharmacological screens and regenerative medicine.
The authors review in some detail the similarities and differences between iPS and ES cells, and what is known about the mechanism of reprogramming. In a commentary about the clinical applications of iPS cells they state that the ability of iPS cells to generate all lineages of the embryo and to contribute to chimera formation lead them to conclude that iPS cells have a developmental potency comparable to ES cells.
- Cell therapy for ischaemic heart disease. Beeres SL, Atsma DE, van Ramshorst J, Schalij MJ, Bax JJ. Heart. 2008;94:1214-26. Review.
This comprehensive review provides an overview of the basic principles of cardiac cell therapy. The basic mechanisms through which cell therapy may improve cardiac performance and the different cell populations that have been tested in preclinical studies are discussed. Also, the different routes of cell delivery are reviewed, along with the results of the currently available clinical studies investigating the safety, feasibility and efficacy of cardiac cell therapy for patients with ischemic heart disease.
Cell types considered are hematopoietic stem cells derived from the BM, endothelial progenitor cells, mesenchymal stem cells, multipotent adult progenitor cells, skeletal myoblasts, resident cardiac progenitor cells, adipose tissue derived cells , umbilical cord derived cells and embryonic stem cells.
Cell delivery routes considered include I.V. infusion, intracoronary cell infusion, and intramyocardial cell injection.
Four tables list and provide references for results of clinical studies of BM transplantation for acute myocardial infarction, BM transplantation for chronic myocardial infarction, skeletal myoblast transplantation for chronic myocardial infarction and BM transplantation for chronic myocardial ischemia.
The authors summarize present findings of clinical studies by saying that randomized studies have produced conflicting data regarding the efficacy of BM transplantation for cardiac repair. A meta-analysis concluded that BM cell transplantation was associated with modest improvements in LVEF and infarct size beyond those achieved with conventional therapy. The authors state that the modest improvements and the favorable safety profile justify the performance of additional randomized, placebo controlled studies. Finally it remains to be assessed whether cell therapy may reduce cardiovascular morbidity and mortality.
- Cell Therapy of Acute Myocardial Infarction: Open Questions. Dimmeler S, Zeiher AM. Cardiology. 2008;113:155-160.
Cell-based therapy is a promising option for treatment of cardiovascular diseases. So far, clinical studies preferentially used adult bone marrow-derived cells for the treatment of patients with acute myocardial infarction. In this article, the authors discuss the results of clinical trials using BM cells for acute and chronic ischemia. They discuss which patients benefit from cell therapy, the timing of cell delivery, the safety of cell therapy, long-term benefit of cell therapy, the mechanism of action and limitations of cell-based therapy.
They indicate that the results of recent cell therapy trials have been criticized for the modest improvement of ejection fraction (meta-analyses: 3 to 3.66%). Larger improvements have been detected in patients with larger myocardial infarcts, i.e., 7.5%.
Attempts of regenerative therapeutic interventions in patients with significant cardiac dysfunction should proceed in controlled trials with the utmost rigorous scientific and ethical standards, paralleled by further extensive in vitro and animal studies.
- Adult stem cells for spinal cord injury: what types and how do they work? Uccelli A. Cytotherapy. 2008;10:541-542.
The author points out that a large number of stem cell-based pre-clinical studies have generated enthusiasm and hope for the treatment of spinal cord injury (SCI). Recent studies reinforce that enthusiasm. One such study is an uncontrolled pilot study showing that transplantation of autologous BM-derived hematopoietic stem cells in 9 patients who had suffered SCI for at least 6 months before treatment, was safe and led to a significant improvement in clinical conditions at 1 year following surgery.
However, such studies do not elucidate the mechanisms behind the clinical improvement. Despite some evidence of neuronal differentiation by injected stem cells, several studies have challenged the biologic relevance of adult stem cell in vivo transdifferentiation into neurons. In contrast, other mechanisms may be more relevant, including bystander effects acting through the release of anti-inflammatory, anti-apoptotic and trophic molecules, and possibly the recruitment of local progenitors by injected stem cells upon migration at the site of injury. Future studies will have to address which stem cell populations, embryonic, mesenchymal, hematopoietic, neural and olfactory ensheathing, among others, may provide the most beneficial effects for CNS repair.
- The significant cardiomyogenic potential of human umbilical cord blood-derived mesenchymal stem cells in vitro. Nishiyama N, Miyoshi S, Hida N, Uyama T, Okamoto K, Ikegami Y, Miyado K, Segawa K, Terai M, Sakamoto M, Ogawa S, Umezawa A. Stem Cells. 2007;25:2017-2024.
Umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are potentially a new cell source for stem cell-based therapy. In this study, human UCBMSCs (5 x 10(3) per cm(2)) were cocultured with fetal murine cardiomyocytes ([CM] 1 x 10(5) per cm(2)). On day 5 of cocultivation, approximately half of the green fluorescent protein (GFP)-labeled UCBMSCs contracted rhythmically and synchronously, suggesting the presence of electrical communication between the UCBMSCs. The fractional shortening of the contracted UCBMSCs was 6.5% +/- 0.7% (n = 20). The UCBMSC-derived cardiomyocytes stained positive for cardiac troponin-I (clear striation +) and connexin 43 (diffuse dot-like staining at the margin of the cell) by the immunocytochemical method. Cardiac troponin-I positive cardiomyocytes accounted for 45% +/- 3% of GFP-labeled UCBMSCs. The cardiomyocyte-specific long action potential duration (186 +/- 12 milliseconds) was recorded with a glass microelectrode from the GFP-labeled UCBMSCs. Cardiomyocytes (CM) were observed in UCBMSCs, which were cocultivated in the same dish with mouse cardiomyocytes separated by a collagen membrane. Cell fusion, therefore, was not a major cause of CM in the UCBMSCs. Approximately half of the human UCBMSCs were successfully transdifferentiated into cardiomyocytes in vitro.
The authors conclude that UCBMSCs can be a promising cellular source for cardiac stem cell-based therapy.
- Neuronal conditioning medium and nerve growth factor induce neuronal differentiation of collagen-adherent progenitors derived from human umbilical cord blood. Arien-Zakay H, Nagler A, Galski H, Lazarovici P. J Mol Neurosci. 2007;32:179-91.
Research in the field of neuronal progenitors is rapidly advancing, driven by the potential use of these cells for cellular therapy of neurodegenerative disorders, stroke, trauma and spinal cord injuries. Since the availability of human neuronal stem cells derived from early embryos is extremely limited, progenitors of other origins are being considered. Human umbilical cord blood (HUCB), has served as an alternative source of hematopoietic stem cells. The biological properties of HUCB attest to the existence of progenitors of multi-lineage capacity, similar to those of BM-derived mesenchymal stem cells. Exposure of these HUCB progenitors to non-selective differentiating agents, such as retinoic acid, DMSO, and betamercaptoethanol, induce properties typical of neuroectodermal-derived cells. The HUCB-derived progenitors may prove useful in a variety of cell-based therapies, including neurological disorders.
The aim of the study was to isolate and characterize a population of neuronal progenitors in HUCB mononuclear cell (MNC) fraction, for in vitro manipulation towards neuronal differentiation. Selection of the HUCB neuronal progenitors (HUCBNPs) was based on the neuronal prerequisite for adherence to collagen. Populations of collagen-adherent, nestin-positive (94.8+/-2.9%) progenitors expressing alpha1/2 integrin receptors, as revealed by Western blot and adhesion assay using selective antagonists, were isolated and survived for more than 14 days. In vitro differentiation of the HUCBNPs was achieved by treatment with 10% human SH-SY5Y neuroblastoma cell-conditioning media (CM) supplemented with 10 ng/ml nerve growth factor (NGF). Some 83+/-8.2% of the surviving progenitors acquired a neuronal-like morphology, expressed by cellular outgrowths of different lengths. About 35+/-6% of the HUCBNPs had long outgrowths with a length/cell diameter ratio greater than 2, typical of developing neurons.
The majority of these progenitors, analyzed by immunocytochemistry and/or RT-PCR, expressed common neuronal markers such as microtubule-associated protein 2 (MAP-2; 98.5+/-2%), neurotrophin receptor (TrkA; 98.5+/-0.06%), neurofillament-160 (NF-160; 94.2+/-1%), beta-tubulin III (89.8+/-4.2%) and neuron specific enolase (NSE). Combined CM and NGF treatment induced constitutive activation of the mitogen-activated protein kinases ERK2 (36-fold vs control), p38alpha (nine-fold vs control) and p38beta (23-fold vs control), most likely related to survival and/or differentiation.
The results point to operationally defined conditions for activating neuronal differentiation of HUCBNPs ex vivo and emphasize the crucial role of neuronal CM and NGF in this process.
