Cellular replacement and regenerative medicine therapies in ischemic stroke.

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.

Autologous infusion of expanded mobilized adult bone marrow-derived CD34+ cells into patients with alcoholic liver cirrhosis.

OBJECTIVES: 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. METHODS: 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. RESULTS: 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 (P < 0.05) 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 (P < 0.05) 1 wk post infusion. The Child-Pugh score improved in 7 out of 9 patients, while 5 patients had improvement in ascites on imaging. CONCLUSION: 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.

High frequency of fetal cells within a primitive stem cell population in maternal blood.

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.

Characterization and clinical application of human CD34+ stem/progenitor cell populations mobilized into the blood by granulocyte colony-stimulating factor.

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.

Cell culture medium composition and translational adult bone marrow-derived stem cell research.

The growing body of adult bone marrow-derived stem cell research in regenerative medicine has recently entered the clinical transplantation setting. The need for a robust flow of data from basic to translational scientists is imperative. All animal- and/or human-derived products should ideally be excluded and synthetic recombinant alternatives used instead. A variety of reasons make such an approach favorable, including achievement of sounder control levels, reduction of any prion, viral, or zoonose contamination risk, avoidance of possible immunological reactions, as well as decrease in demand on animal supplies. At a preclinical level, investigative cell culture protocols must incorporate serum-free media groups alongside control conditions. Finally, autologous serum should not be neglected until an "off-the-shelf" alternative becomes available in the future.

Selective suppression of cathepsin L by antisense cDNA impairs human brain tumor cell invasion in vitro and promotes apoptosis.

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.

Lysosomal enzymes, cathepsins in brain tumour invasion.

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.

Comparison of potential biological markers cathepsin B, cathepsin L, stefin A and stefin B with urokinase and plasminogen activator inhibitor-1 and clinicopathological data of breast carcinoma patients.

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.