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Worldwide, tissue engineering and cellular replacement therapies are at the forefront of the regenerative medicine agenda, and researchers are addressing key diseases, including diabetes, stroke and neurological disorders. It is becoming evident that neurological cell therapy is a necessarily complex endeavor. The brain as a cellular environment is complex, with diverse cell populations, including specialized neurons (e.g., dopaminergic, motor and glutamatergic neurons), each with specific functions. The population also contains glial cells (astrocytes and oligodendrocytes) that offer the supportive network for neuronal function. Neurological disorders have wide and varied pathologies; they can affect predominantly one cell type or a multitude of cell types, which is the case for ischemic stroke. Both neuronal and glial cells are affected by stroke and, depending on the region of the brain affected, different specialized cells are influenced. This review will address currently available therapies and focus on the application and potential of cell replacement, including stem cells and immortalized cell line-derived neurons as regenerative therapies for ischemic stroke, addressing current advances and challenges ahead.

Recent advances in regenerative medicine, including hematopoietic stem cell (HSC) transplantation, have brought hope for patients with severe alcoholic liver cirrhosis (ALC). The aim of this study was to assess the safety and efficacy of administering autologous expanded mobilized adult progenitor CD34+ cells into the hepatic artery of ALC patients and the potential improvement in the liver function.Nine patients with biopsy-proven ALC, who had abstained from alcohol for at least 6 months, were recruited into the study. Following granulocyte colony-stimulating factor (G-CSF) mobilization and leukapheresis, the autologous CD34+ cells were expanded in vitro and injected into the hepatic artery. All patients were monitored for side effects, toxicities, and changes in the clinical, hematological, and biochemical parameters.On average, a five-fold expansion in cell number was achieved in vitro, with a mean total nucleated cell count (TNCC) of 2.3 x 10(8) pre infusion. All patients tolerated the procedure well, and there were no treatment-related side effects or toxicities observed. There were significant decreases in serum bilirubin (P0.05) 4, 8, and 12 wk post infusion. The levels of alanine transaminase (ALT) and aspartate transaminase (AST) showed improvement through the study period and were significant (P0.05) 1 wk post infusion. The Child-Pugh score improved in 7 out of 9 patients, while 5 patients had improvement in ascites on imaging.It is safe to mobilize, expand, and reinfuse autologous CD34+ cells in patients with ALC. The clinical and biochemical improvement in the study group is encouraging and warrants further clinical trials.

Evidence is growing in support of the role of stem cells as an attractive alternative in treatment of liver diseases. Recently, we have demonstrated the feasibility and safety of infusing CD34(+) adult stem cells; this was performed on five patients with chronic liver disease. Here, we present the results of long-term follow-up of these patients. Between 1 x 10(6) and 2 x 10(8) CD34(+) cells were isolated and injected into the portal vein or hepatic artery. The patients were monitored for side effects, toxicity and changes in clinical, haematological and biochemical parameters; they were followed up for 12-18 months. All patients tolerated the treatment protocol well without any complications or side effects related to the procedure, also there were no side effects noted on long-term follow-up. Four patients showed an initial improvement in serum bilirubin level, which was maintained for up to 6 months. There was marginal increase in serum bilirubin in three of the patients at 12 months, while the fourth patient's serum bilirubin increased only at 18 months post-infusion. Computed tomography scan and serum alpha-foetoprotein monitoring showed absence of focal lesions. The study indicated that the stem cell product used was safe in the short and over long term, by absence of tumour formation. The investigation also illustrated that the beneficial effect seemed to last for around 12 months. This trial shows that stem cell therapy may have potential as a possible future therapeutic protocol in liver regeneration.

Summary Recent developments in the field of regenerative stem cell therapy for ischaemic heart disease have lead to an explosion in the clinical trial realm. At present no consensus exists regarding, amongst others, the optimal cell type as well as the underlying mechanism of action for any clinical improvement observed. As de novo reconstitution of myocardial tissue from multipotent stem cells is one of the working theories, the transdifferentiation potential of cellular populations under investigation into cardiomyocyte lineage phenotypes must ideally be assessed in preclinical bench work. Culture medium composition and a variety of growth factors are crucial determinants in this process. We discuss all relevant data acquired from in vitro work.

A phase I study was performed to determine the safety and tolerability of injecting autologous CD34(+) cells into five patients with liver insufficiency. The study was based on the hypothesis that the CD34(+) cell population in granulocyte colony-stimulating factor (G-CSF)-mobilized blood contains a subpopulation of cells with the potential for regenerating damaged tissue. We separated a candidate CD34(+) stem cell population from the majority of the CD34(+) cells (99%) by adherence to tissue culture plastic. The adherent and nonadherent CD34(+) cells were distinct in morphology, immunophenotype, and gene expression profile. Reverse transcription-polymerase chain reaction-based gene expression analysis indicated that the adherent CD34(+) cells had the potential to express determinants consistent with liver, pancreas, heart, muscle, and nerve cell differentiation as well as hematopoiesis. Overall, the characteristics of the adherent CD34(+) cells identify them as a separate putative stem/progenitor cell population. In culture, they produced a population of cells exhibiting diverse morphologies and expressing genes corresponding to multiple tissue types. Encouraged by this evidence that the CD34(+) cell population contains cells with the potential to form hepatocyte-like cells, we gave G-CSF to five patients with liver insufficiency to mobilize their stem cells for collection by leukapheresis. Between 1 x 10(6) and 2 x 10(8) CD34(+) cells were injected into the portal vein (three patients) or hepatic artery (two patients). No complications or specific side effects related to the procedure were observed. Three of the five patients showed improvement in serum bilirubin and four of five in serum albumin. These observations warrant further clinical trials.

Invasion and metastasis of certain tumors are accompanied by increased mRNA protein levels and enzymatic activity of cathepsin L. Cathepsin L has also been suggested to play a role in the proteolytic cascades associated with apoptosis. To investigate the role of cathepsin L in brain tumor invasion and apoptosis, the human glioma cell line, IPTP, was stably transfected with full-length antisense and sense cDNA of cathepsin L. Down-regulation of cathepsin L by antisense cDNA significantly impaired (up to 70%) glioma cell invasion in vitro and markedly increased glioma cell apoptosis induced by staurosporine. Compared to control and parental cell lines, antisense down-regulation of cathepsin L was associated with an earlier induction of caspase-3 activity. Up-regulation of cathepsin L activity by sense cDNA was associated with reduced apoptosis and later induction of caspase-3 activity. Moreover, down-regulation of cathepsin L lowered the expression of antiapoptotic protein Bcl-2, whereas up-regulation increased the expression of Bcl-2, indicating that cathepsin L acts upstream of caspase-3. These data show that cathepsin L is an important protein mediating the malignancy of gliomas and its inhibition may diminish their invasion and lead to increased tumor cell apoptosis by reducing apoptotic threshold.

The expression patterns of different classes of peptidases in central nervous system (CNS) tumours have been most extensively studied in astrocytomas and meningiomas. Although the two types of tumours are very different in most respects, both may invade locally into normal brain. This process of invasion includes increased synthesis and secretion of lysosomal proteolytic enzymes – cathepsins. Aspartic endopeptidase cathepsin (Cat) D levels were found to be elevated in high-grade astrocytoma and partial inhibition of glioblastoma cell invasion by anti-Cat D antibody suggests that the enzyme activity is involved in the invasion process. Several studies on cysteine endopeptidase (CP) Cat B in gliomas agreed that transcript abundance, protein level and activity of Cat B increased in high-grade astrocytoma cultures compared with low-grade astrocytoma cultures and normal brain. Moreover, in glioma biopsies Cat B levels correlated with evidence of clinical invasion and it has been demonstrated that Cat B both in tumour cells and in endothelial cells can serve as a new biological marker for prognosis in glioblastoma patients. A high level of Cat B protein was also a diagnostic marker for invasive types of meningioma, distinguishing between histomorphologically benign, but invasive meningiomas and noninvasive, so-called clear–benign meningiomas. Cat L was also significantly increased in high-grade astrocytoma compared with low-grade astrocytoma and normal brain. Specific Cat L antibodies and antisense Cat L RNA transfection significantly lowered glioblastoma cell invasion. In meningioma, Cat L was a less-significant marker of invasion than Cat B. In contrast to cathepsins, the activities of endogenous cysteine peptidase inhibitors (CPIs), including stefins, cystatins and kininogens, were significantly higher in benign and atypical meningioma cell extracts than in malignant meningioma, and low-grade compared to high-grade astrocytoma. However, very low levels of stefins A and B were found in meningioma and glioblastoma tissues. Further studies on the expression levels and balance between cysteine endopeptidases (CPs) and CPIs would improve the clinical application of cathepsins in prognosis, which would lead to more-informed therapeutic strategies.

The idea behind cell therapy is not new; it was proposed many years ago. However, the significant research activities in the regenerative medicine field started in the 1970s. Over the last decade the advances in the understanding of stem cell biology have made the prospect of tissue regeneration a potential clinical reality. A potential strategy to replace, repair and restore the function of damaged tissues or organs is stem cell transplantation. Cell therapeutics may perform better than current treatments such as medical devices, recombinant proteins and chemical compounds. Despite advances made in the past few years and the accumulating body of evidence supporting the contribution of stem cells in tissue regeneration, many questions regarding their clinical application need to be addressed [1]. Processes to produce cell-based therapeutics are not standardized yet and are highly variable. The critical challenge for translating stem cell therapy into the clinic is to deliver safe, effective and affordable therapies to patients. Types of cells and routes of administration for specific clinical scenarios would need to be determined in order to maximize potential therapeutic benefit. Furthermore, it is necessary to determine the quantity of cells needed for transplantation to achieve best possible therapeutic effect. The type of patients that would benefit from this treatment must be determined. The desirable features of stem cells for therapy include rapid expansion in vitro and minimal immunogenicity and regenerative properties when transplanted into injured organs. Possible candidate cells to be used include various stem cells such as adult stem cells, embryonic stem (ES) cells and recently generated pluripotent stem cells (iPS) [2] that closely resemble ES cells. ES cells have an unlimited self-renewal ability and the capacity to differentiate into any specialized cell type, therefore, could represent an unlimited cell source for tissue regeneration. However, research on these cells has been hampered or banned in some countries because of the ethical concerns about destroying human embryos to obtain them. Another major limiting factor for their usefulness in clinical therapy lies in their risk of uncontrolled growth and potential danger of teratoma formation and immunological intolerance [3,4]. In spite of this, the US biotechnology company Geron have performed the first US FDA-approved clinical trial using ES-derived glial oligodendrocytes in patients with acute spinal cord injury. The results have not been published yet, but the trial is aiming to elucidate whether ES cells are safe to use in patients and also if they improve patients’ sensation in the trunk or legs. Using iPS cells can bypass ethical and immunological issues and may offer the possibility of developing patient-specific cell therapeutics. However, the use of oncogenic viruses to make the iPS is considered unsafe for clinical applications and there is a possibility of tumour genesis. Improved induction methods more suitable for clinical use are under development [5]. Moreover, patient-specific therapy is associated with high cost and regulatory issues. Currently

Cancer is a disease of the gene, caused by genetic and epigenetic mutations, resulting in the development of malignant cells. Previous concepts of the monoclonal origin of tumors and the stochastic nature of carcinogenesis are being challenged by hierarchical/plasticity models that predict the existence of tumor-initiating and tumor-propagating cells, which have stem cell properties and are thus highly resistant to therapy. It may therefore be postulated that cancer is a stem cell disease. One of the actively investigated tumors of cancer stem cell origin is glioma, one of the commonest primary brain tumors in adults. In this chapter we shall focus on glioblastoma multiforme (GBM), glioma’s most malignant stage, characterized by enhanced, single-cell infiltration into normal brain. The invasive cell phenotype is the major obstacle for successful therapy, as highly invasive cells that can migrate from the bulk tumor mass cannot be removed by any currently available therapy. Invasion is a complex, stepwise process, initiated at various levels—from gene activation to the level of protein post-translational modification. At the phenotype level, the process is reminiscent of the epithelial to mesenchymal transition in carcinoma malignization, which includes the activation of the so-called migratome, possibly overlapping with proteases, i.e. a cancer degradome. On the basis of the hierarchical/plasticity model of carcinogenesis, the cells undergoing epithelial to mesenchymal transition may also have stem/progenitor cell characteristics. Other types of stem cells associated with tumors are infiltrating haematopoietic stem cells, endothelial progenitor cells and mesenchymal stem cells (MSCs), comprising the tumor microenvironment. Their role and differentiation pathways in tumor niches are the subject of intensive investigations, and unravelling the mechanisms underlying MSC-mediated modulation of tumor behaviour via paracrine and direct interactions with GBM stem cells in vitro may solve the dispute regarding their antimalignant activity, which is currently contradictory to the hypothesis of a possible mesenchymal origin of GBM stem cells. By the same token, MSCs may be novel therapeutic vectors for drug delivery, which are badly needed. Cell therapies using stem cells are promising owing to their selective tropism towards tumors. In particular, autologous MSCs are considered promising at least in some tumors, especially owing to their immunomodulatory potential. However, MSC therapy may have adverse effects, since the role of MSCs in tumor progression has been evidenced. Combined cell-based drug and gene delivery with anti-invasive strategies could therefore contribute to enhanced efficacy of conventional therapeutic approaches in glioblastoma treatment. In this chapter we shall thoroughly revisit the advantages of and obstacles in recent preclinical trials using stem cells for treating brain tumors.

The existence of cancer stem cells continues to be extensively debated among scientists, and given the mounting evidence, it is an area that needs further examination. The possible reason for resistance to current treatment modalities in cancer is that we have yet to discover a method to eradicate cancer stem cells. This chapter describes the theory of cancer stem cells, portrays the role of cancer stem cells in radiation and chemotherapy resistance, and describes methods for their isolation. Finally, it presents current knowledge on cancer stem cells in solid organ tumors and discusses the limitations of current isolation strategies.

This paper highlights our experience of the transfer of hydrodynamic gene therapy (HGT) from the large animal, the pig, into clinical practice. The modification of balloon catheters and the development of a minimally invasive technique to allow selective isolation of liver segments for HGT in the large animal and human are described. Finally, our preliminary results from a phase I clinical study of HGT for thrombopoietin (TPO) in cirrhotic patients with thrombocytopenia are discussed. Based on these provisional data, minimally invasive selective HGT of liver segments appears to be technically safe, but further work is required to optimize the efficiency of gene transfer in order to achieve clinical benefit.

BACKGROUND: During pregnancy, fetal cells enter the maternal bloodstream resulting in fetal cell microchimerism. The fetal cells persist in the mother for decades and colonize a variety of maternal organs. They are associated with maternal autoimmune diseases and may also participate in tissue repair. The identity of the microchimeric cells is not certain but they must be able to persist long-term and have potential for multitissue differentiation. METHODS AND RESULTS: Here we tested the hypothesis that the fetal microchimeric cells are primitive stem cells, represented by CD34 + adherent cells, which have a wide potential for differentiation. We isolated these stem cells from the blood of pregnant females (n = 25) and detected fetal cells of the correct gender, using fluorescence in situ hybridization, in a high proportion (71% male fetuses and 90% female fetuses; false positive rate 11%, false negative rate 29%) of cases. By RT-PCR, we demonstrated that the cells express Oct-4, Nanog and Rex-1. No fetal cells were detected in the mononuclear or total CD34 + cell populations but high frequencies (mean 11.8 %) of fetal cells were detected in the adherent CD34+ cell population. CONCLUSIONS: These results identify adherent CD34+ stem cells as candidate fetal microchimeric cells, which are capable of sustaining the fetal cell population in the long term and have the ability to colonize multiple tissues and organs.

The gene therapy dream started well over a decade ago. Despite its wide and successful application in research, it still has not reached the clinics in a meaningful way. No doubt that successful gene therapy has a lot to offer to patients with inherited, benign or malignant diseases

Organ transplantation currently remains the definitive cure for several liver and pancreatic disorders. However, due to a major shortage of available organs many are turning to alternative sources for whole organ or cell-based transplantation. Stem cells have been proposed as an alternative to organ transplantation. The ethical and legal issues associated with embryonic stem cells have shifted the focus to adult stem cells and their regenerative potential has been under intense investigation. In this chapter we are reviewing the latest advances in stem cell therapy in liver diseases from bench to bedside.

Advances in stem cell biology and the discovery of pluripotent stem cells have made the prospect of cell therapy and tissue regeneration a clinical reality. Cell therapies hold great promise to repair, restore, replace or regenerate affected organs and may perform better than any pharmacological or mechanical device. There is an accumulating body of evidence supporting the contribution of adult stem cells, in particular those of bone marrow origin, to liver and pancreatic islet cell regeneration. In this review, we will focus on the cell therapy for the diseased liver and pancreas by adult haematopoietic stem cells, as well as their possible contribution and application to tissue regeneration. Furthermore, recent progress in the generation, culture and targeted differentiation of human haematopoietic stem cells to hepatic and pancreatic lineages will be discussed. We will also explore the possibility that stem cell technology may lead to the development of clinical modalities for human liver disease and diabetes.

Defining Progenitors Based on Their Expression of Aldehyde Dehydrogenase (R W Storms) Fetal Mesenchymal Stem Cells Are More Primitive Than Adult Mesenchymal Stem Cells (C Gotherstrom et al.) The Immunoregulatory Role of Mesenchymal Stem Cells (F Dazzi & J Timoshanko) Understanding Cell Migration Through the Paradigm of T-Lymphocyte Homing (V Mirenda & F M Marelli-Berg) Blueprint for the Response of Blood and Bone Marrow-Derived Stem Cells and Their Progeny to Hypoxia (S M Watt et al.) The Potential of Stem Cells in Tissue Engineering (N D Evans & J M Polak) Joint Tissue Engineering (C De Bari et al.) Adult Stem Cells as a Treatment for Liver Diseases (N Levicar) The Generation of Pancreatic Beta Cells from Stem Cells: Intra- and Extra-Pancreatic Sources (M Brittan et al.) Cytokines and Stem Cell Differentiation into Cardiomyocyte Lineage Cells (I Dimarakis) Regulatory Networks Controlling Neural Stem Cell Self-Renewal and Differentiation (Y Shi) Demyelination as a Therapeutic Target in Spinal Cord Injury (J R Faulkner & H S Keirstead) Microchimeric Fetal Stem Cells and Non-Invasive Prenatal Genetic Diagnosis (M A A Mikhail) The Role of Stem Cells in Liver and Gastrointestinal Cancer (M R Alison et al.) Embryonic Stem Cells: Innovation in Predictive Toxicology (G G Cezar & J Quam).

Cysteine, serine and metalloproteinases and their respective inhibitors are involved in tumor cell invasion and may have prognostic value for the outcome of malignant disease. The aim of the study was to compare the expression of new potential biological tumor markers, the lysosomal cysteine proteinases and their endogenous inhibitors, with that of the serine proteinases and their inhibitors in breast cancinoma and to relate their levels to the clinicopathological factors of the disease. Enzyme-linked immunosorbent assays (ELISAs) were used to measure cysteine cathepsin B (CatB) and cathepsin L (CatL) and their inhibitors, stefin A (StA) and stefin B (StB), together with urokinase (u-PA) and plasminogen activator inhibitor-1 (PAI-1), in 150 cytosols of primary invasive breast carcinoma. A good correlation was found between the levels of the two cysteine proteinases but only a moderate one between those of the cysteine and serine proteinases. u-PA and PAI-1 levels correlated positively with histological grade and negatively with estrogen receptor (ER) status. PAI-1 correlated with most clinicopathological factors that indicate the progression of the disease, while cathepsins and stefins were independent of these factors. In the total group of patients, high u-PA and PAI-1 and low StB levels correlated significantly with shorter disease-free survival (DFS), while CatB, CatL and StA did not. In lymph node negative patients, high CatB and CatL were also associated with shorter DFS, while u-PA remained the most significant of all these biological markers. In conclusion, this retrospective study showed u-PA to be of better prognostic relevance than the cysteine proteinases, though CatB and CatL were relevant for prognosis in lymph node negative breast cancer patients.