Grafting dopamine neurons in Parkinson's disease: do stem cells have a role in the future?

Previous studies have suggested that human umbilical cord blood (HUCB) may serve as a rich source of hematopoietic and nonhematopoietic stem cells and that conditions exist that can coax hematopoietic cells to express neural characteristics. In our laboratory, these cells were tested for several models of neurodegenerative diseases and spinal cord injuries. Through a series of transplantation studies we have begun to uncover the properties of HUCB-derived cells in neuropoietic regions of the neonatal (1) and aging rodent brain. The systematic application of phenotyping approaches to characterize survival, migratory potential and morphologic properties of the differentiated HUCB progeny within normal/unaffected brain will serve as a base for understanding the potential effect of these cells in the diseased brain.
Acknowledgements:  Supported in part by R01 6155039 (TZ). HUCB cells were obtained from Saneron CCEL Therapeutics, Inc. and BioWhittaker, Inc.     

Mannitol facilitates neurotrophic factor up‐regulation and behavioural recovery in neonatal hypoxic‐ischaemic rats with human umbilical cord blood grafts

We recently demonstrated that blood–brain barrier permeabilization using mannitol enhances the therapeutic efficacy of systemically administered human umbilical cord blood (HUCB) by facilitating the entry of neurotrophic factors from the periphery into the adult stroke brain. Here, we examined whether the same blood–brain barrier manipulation approach increases the therapeutic effects of intravenously delivered HUCB in a neonatal hypoxic‐ischaemic (HI) injury model. Seven‐day‐old Sprague–Dawley rats were subjected to unilateral HI injury and then at day 7 after the insult, animals intravenously received vehicle alone, mannitol alone, HUCB cells (15k mononuclear fraction) alone or a combination of mannitol and HUCB cells. Behavioural tests at post‐transplantation days 7 and 14 showed that HI animals that received HUCB cells alone or when combined with mannitol were significantly less impaired in motor asymmetry and motor coordination compared with those that received vehicle alone or mannitol alone. Brain tissues from a separate animal cohort from the four treatment conditions were processed for enzyme‐linked immunosorbent assay at day 3 post‐transplantation, and revealed elevated levels of GDNF, NGF and BDNF in those that received HUCB cells alone or when combined with mannitol compared with those that received vehicle or mannitol alone, with the combined HUCB cells and mannitol exhibiting the most robust neurotropic factor up‐regulation. Histological assays revealed only sporadic detection of HUCB cells, suggesting that the trophic factor–mediated mechanism, rather than cell replacement per se, principally contributed to the behavioural improvement. These findings extend the utility of blood–brain barrier permeabilization in facilitating cell therapy for treating neonatal HI injury.     

Transplantation of human umbilical cord blood cells improves glycemia and glomerular hypertrophy in type 2 diabetic mice

Recent in vitro and in vivo studies have shown that either animal- or human-derived embryonic stem cells can differentiate into insulin-secreting cells and lower blood glucose levels. However, studies utilizing human umbilical cord blood (HUCB) mononuclear cells to improve blood glucose levels in diabetic animals have received little attention. In this study, we examined the effect of transplanted HUCB mononuclear cells on blood glucose levels, survival, and renal pathology in obese mice with spontaneous development of type 2 diabetes. The results show that injection of HUCB mononuclear cells into orbital plexus of mice caused improvement not only in blood glucose levels and survival rate but also normalization of glomerular hypertrophy and tubular dilatation. Thus, transplantation of HUCB mononuclear cells appears to be another modality of stem cell therapy in diabetes mellitus.     

Phenotypical and functional properties of human bone marrow mesenchymal progenitor cells

Bone marrow stroma provides the microenvironment for hematopoiesis and is also the source of mesenchymal progenitors (mesenchymal or marrow stromal cells [MSC]) that may serve as long-lasting precursors for bone, cartilage, lung, and muscle. While several studies have indicated the differentiation potential of MSC, few studies have been performed on the cells themselves. In an attempt to further expand our knowledge on these cells, we have performed studies on their cell cycle, immuno- and adhesive-phenotype, ex vivo expansion, and differentiation properties. MSC cultures have been initiated from human bone marrow low-density mononuclear cells and maintained in the absence of differentiation stimuli and hematopoietic cells. The homogenous layer of adherent cells thus formed exhibits a typical fibroblastlike morphology, a population doubling time of 33 h, a large expansive potential, and cell cycle characteristics including a subset (20%) of quiescent cells. The antigenic phenotype of MSC is not unique, borrowing features of mesenchymal, endothelial, and epithelial cells. Together, MSC express several adhesion-related antigens, like the integrin subunits α4, α5, β1, integrins αvβ3 and αvβ5, ICAM-1, and CD44H. MSC produce and functionally adhere to extracellular matrix molecules. When incubated under proper stimuli, MSC differentiate into osteoblasts or adipocytes. Taken together, these results demonstrate that adherent marrow-derived cells cultured in the absence of hematopoietic cells and differentiation stimulus give rise to a population of cells with phenotypical and functional features of mesenchymal progenitors. The existence of a subset of quiescent cells in MSC cultures seems to be extremely significant, since their number and properties should be enough to sustain a steady supply of cells that upon proliferation and commitment may serve as precursors for a number of nonhematopoietic tissues. J. Cell. Physiol. 181:67–73, 1999. © 1999 Wiley-Liss, Inc.     

Human umbilical cord blood effect on sod mice (amyotrophic lateral sclerosis)

In previous studies we observed that human umbilical cord blood (HUCB) could have a protective effect on the onset of disease and time of death in MRL Lpr/Lpr mice which have an autoimmune disease that may be considered similar to human lupus. We believed a temporary xenograph may have occurred in these animals with the disease process delayed and the life span markedly increased. When HUCB is stored at 4 degrees C in gas permeable bags, there is a decrease of the cell reaction in mixed lymphocyte cultures. The blood, however, maintains a significant number of cells capable of producing replatable colonies. This study attempted to determine the effect of HUCB on SOD1 mice (transgenic B6SJL-TgN(SOD1-G93A)1GUR), which have a mutation of the human transgene, (CuZn superoxide dismutase gene SOD1) that has been associated with amyotrophic lateral sclerosis. We previously developed evidence that the survival of lethally irradiated mice was related to the number of human mononuclear cells administered. In the present study, we decided to investigate the effect of a relatively large dose of human mononuclear cord blood cells on SOD1 mice subjected to a sublethal dose of irradiation preceded by antikiller sera (rabbit anti-asialo). The SOD1 mice show evidence of paralysis at 4 to 5 months. The average expected lifetime of these mice is reported to be 130 days (Jackson Laboratory). In this experiment, there were 23 mice. Two mice died before the onset of paralysis. The remainder were divided into three groups: group I: control group of 4 untreated mice; group II: an experimental group of 6 mice treated with antikiller sera, 800 cGy irradiation plus 5 x 10(6) congenic bone marrow mononuclear cells; group III: another experimental group of 11 mice treated with antikiller sera, 800 cGy irradiation plus 34.2-35.6 x 10(6) HUCB mononuclear cells, previously stored for 17-20 days at 4 degrees C in gas permeable bags. The results were as follows: the average age at death was: (I) 127 days for the untreated control group, (II) 138 days for the group that received 800 cGy of irradiation and congenic bone marrow (BM) and (III) 148 days for the group that received irradiation and HUCB. (P < 0.001 HUCB vs control, p < 0.01 HUCB vs BM). The longest surviving mouse in each group was 131, 153, and 182 days old respectively. In summary, large doses of HUCB mononuclear cells produced considerable delay in the onset of symptoms and death of SOD1 mice. These preliminary results may not only indicate that amyotrophic lateral sclerosis is an autoimmune disease, but may also indicate a possible treatment for a devastating disease and possibly others.     

Murine survival of lethal irradiation with the use of human umbilical cord blood.

We have found that human umbilical cord blood (HUCB) will routinely protect mice exposed to lethal levels of irradiation. At the end of 50 days, over seventy percent (70%) of mice injected with HUCB survived 900 cGy or irradiation, which produced 100% deaths in the uninjected control animals. Moreover, there was some evidence that human colony stimulating factors further improved survival. Anti-Natural Killer cell (NK) antibody was utilized along with HUCB in these studies, however, Anti-NK cell serum alone had no radioprotective effect in mice. The studies reported here suggest the possibility of utilizing HUCB for immediate protection of humans from lethal irradiation.     

Human umbilical cord blood cells protect oligodendrocytes from brain ischemia through Akt signal transduction

Human umbilical cord blood (HUCB) cells protect the brain against ischemic injury, yet the mechanism of protection remains unclear. Using both in vitro and in vivo paradigms, this study examined the role of Akt signaling and peroxiredoxin 4 expression in human umbilical cord blood cell-mediated protection of oligodendrocytes from ischemic conditions. As previously reported, the addition of HUCB cells to oligodendrocyte cultures prior to oxygen glucose deprivation significantly enhanced oligodendrocyte survival. The presence of human umbilical cord blood cells also increased Akt phosphorylation and elevated peroxiredoxin 4 expression in oligodendrocytes. Blocking either Akt or peroxiredoxin 4 activity with Akt Inhibitor IV or a peroxiredoxin 4-neutralizing antibody, respectively, negated the protective effects of human umbilical cord blood cells. In vivo, systemic administration of human umbilical cord blood cells 48 h after middle cerebral artery occlusion increased Akt phosphorylation and peroxiredoxin 4 protein expression while reducing proteolytic cleavage of caspase 3 in oligodendrocytes residing in the ipsilateral external capsule. Moreover, human umbilical cord blood cells protected striatal white matter bundles from degeneration following middle cerebral artery occlusion. These results suggest that the soluble factors released from human umbilical cord blood cells converge on Akt to elevate peroxiredoxin 4 levels, and these effects contribute to oligodendrocyte survival.     

Stem cells from umbilical cord blood differentiate into myotubes and express dystrophin in vitro only after exposure to in vivo muscle environment

BACKGROUND INFORMATION: Duchenne muscular dystrophy is a disease characterized by progressive and irreversible muscle degeneration for which there is no therapy. HUCB (human umbilical cord blood) has been considered as an important source of haematopoietic and mesenchymal stem cells, each having been shown to differentiate into distinct cell types. However, it remains unclear if these cells are able to differentiate into muscle cells. RESULTS: We have showed that stem cells from HUCB did not differentiate into myotubes or express dystrophin when cultured in muscle-conditioned medium or with human muscle cells. However, delivery of GFP (green fluorescent protein)-transduced mononucleated cells from HUCB, which comprises both haematopoietic and mesenchymal populations, into quadriceps muscle of mdx (mouse dystrophy X-chromosome linked) mice resulted in the expression of human myogenic markers. After recovery of these cells from mdx muscle and in vitro cultivation, they were able to fuse and form GFP-positive myotubes that expressed dystrophin. CONCLUSIONS: These results indicate that chemical factors and cell-to-cell contact provided by in vitro conditions were not enough to trigger the differentiation of stem cells into muscle cells. Nevertheless, we showed that the HUCB-derived stem cells were capable of acquiring a muscle phenotype after exposure to an in vivo muscle environment, which was required to activate the differentiation programme.     

Migration, fate and in vivo imaging of adult stem cells in the CNS

Adult stem cells have been intensively studied for their potential use in cell therapies for neurodegenerative diseases, ischemia and traumatic injuries. One of the most promising cell sources for autologous cell transplantation is bone marrow, containing a heterogenous cell population that can be roughly divided into hematopoietic stem and progenitor cells and mesenchymal stem cells (MSCs). MSCs are multipotent progenitor cells that, in the case of severe tissue ischemia or damage, can be attracted to the lesion site, where they can secrete bioactive molecules, either naturally or through genetic engineering. They can also serve as vehicles for delivering therapeutic agents. Mobilized from the marrow, sorted or expanded in culture, MSCs can be delivered to the damaged site by direct or systemic application. In addition, MSCs can be labeled with superparamagnetic nanoparticles that allow in vivo cell imaging. Magnetic resonance imaging (MRI) is thus a suitable method for in vivo cell tracking of transplanted cells in the host organism. This review will focus on cell labeling for MRI and the use of MSCs in experimental and clinical studies for the treatment of brain and spinal cord injuries.     

The repopulating potential and differentiation capacity of hematopoietic stem cells from the blood and bone marrow of normal mice

Using the hematopoietic colony technique, we have investigated the repopulating potential of bone marrow cells and leukocytes of blood from normal mice and have demonstrated that the frequency of hematopoietic stem cells in bone marrow is 50 to 150 times that of stem cells in the circulating blood. The differentiation capacity of these stem cells has also been examined. Results of comparative studies of the serial sections of hematopoietic colonies formed from marrow and blood leukocytes indicate that the differentiation capacity of stem cells from marrow and blood is similar, and that at least 80% of these cells differentiate along a single cell line. Thus, peripheral blood stem cells can effect a complete hematopoietic graft, establishing in the host, donor red cells, granulocytes, and platelets. The possibility that blood leukocytes may serve as a potential source of stem cells for hematopoietic transplants has been considered. Although blood contains stem cells, their frequency is so low as to make it unlikely that they would become a useful source of precursor cells for transplantation purposes.     

Human umbilical cord blood cells transfected with VEGF and L(1)CAM do not differentiate into neurons but transform into vascular endothelial cells and secrete neuro-trophic factors to support neuro-genesis-a novel approach in stem cell therapy

Genetically modified mono-nuclear cell fraction from human umbilical cord blood (HUCB) expressing human vascular endothelial growth factor (VEGF) and mouse neural L₁ cell adhesion molecule (L₁CAM) were used for gene-stem cell therapy of transgenic ^G93^A mice adopted as an animal amyotrophic lateral sclerosis (ALS) model. We generated non-viral plasmid constructs, expressing human VEGF₁₆₅ (pcDNA-VEGF) and mouse neural L₁ cell adhesion molecule (pcDNA-mL₁CAM). Mono-nuclear fraction of HUCB cells were transiently transfected by electro-poration with a mixture of expression plasmids (pcDNA-VEGF + pcDNA-mL₁CAM). Sixteen transgenic female and male mice were randomly assigned to three groups: (1) transplantation of genetically modified HUCB cells expressing L₁ and VEGF (n = 6), (2) transplantation of un-transfected HUCB cells (n = 5), and (3) control group (n = 5). In first two experimental groups 1 × 10⁶ cells were injected retro-orbitally in pre-symptomatic 22–25-week-old ^G93^A mice. Our results demonstrate that HUCB cells successfully grafted into nervous tissue of ALS mice and survived for over 3 months. Therefore, genetically modified HUCB cells migrate in the spinal cord parenchyma, proliferate, but instead of transforming into nerve cells, they differentiate into endothelial cells forming new blood vessels. We propose that: (A) expression of mouse neural L₁CAM is responsible for increased homing and subsequent proliferation of transplanted cells at the site of neuro-degeneration, (B) expression of human VEGF directs HUCB cell differentiation into endothelial cells, and (C) neuro-protective effect may stem from the delivery of various neuro-trophic factors from newly formed blood vessels.     

Neural stem cells—trends and advances

For many years, accepted dogma held that brain is a static organ with no possibility of regeneration of cells in injured or diseased human brain. However, recent preclinical reports have shown regenerative potential of neural stem cells using various injury models. This has resulted in renewed hope for those suffering from spinal cord injury and neural damage. As the potential of stem cell therapy gained impact, these claims, in particular, led to widespread enthusiasm that acute and chronic injury of the nervous system would soon be a problem of the past. The devastation caused by injury or diseases of the brain and spinal cord led to wide premature acceptance that “neural stem cells (NSCs)” derived from embryonic, fetal or adult sources would soon be effective in reversing neural and spinal trauma. However, neural therapy with stem cells has not been realized to its fullest extent. Although, discrete population of regenerative stem cells seems to be present in specific areas of human brain, the function of these cells is unclear. However, similar cells in animals seem to play important role in postnatal growth as well as recovery of neural tissue from injury, anoxia, or disease. J. Cell. Biochem. 114: 764–772, 2013. © 2012 Wiley Periodicals, Inc.     

Human umbilical cord blood-derived cells differentiate into hepatocyte-like cells in the Fas-mediated liver injury model

Human umbilical cord blood (HUCB) contains stem/progenitor cells, which can differentiate into a variety of cell types. In this study, we investigated whether HUCB cells differentiate into hepatocytes in vitro and in vivo. We also examined whether CD34 could be the selection marker of stem cells for hepatocytes. HUCB cells were obtained from normal full-term deliveries, and CD34(+/-) cells were further separated. For in vitro study, HUCB cells were cultured for 4 wk, and expressions of liver-specific genes were examined. For the in vivo study, nonobese diabetic/severe combined immunodeficient mice were subjected to liver injury by a Fas ligand-carried adenoviral vector or only radiated. Mice were treated simultaneously with or without cell transplantation of HUCB, CD34(+), or CD34(-) cells. After 4 wk, human-specific gene/protein expression was examined. In the in vitro study, human liver-specific genes were positive after 7 days of culture. The immunofluorescent study showed positive staining of alpha-fetoprotein, cytokeratin 19, and albumin in round-shaped cells. In the in vivo study, immunohistochemical analysis showed human albumin-positive, hepatocyte-specific antigen-positive cells in mouse livers of the Fas ligand/transplantation group. Fluorescence in situ hybridization analysis using the human Y chromosome also showed positive signals. However, no difference between transplanted cell types was detected. In contrast, immunopositive cells were not detected in the irradiated/transplantation group. The RT-PCR result also showed human hepatocyte-specific gene expressions only in the Fas ligand/transplantation group. HUCB cells differentiated into hepatocyte-like cells in the mouse liver, and liver injury was essential during this process. The differences between CD34(+) and CD34(-) cells were not observed in human hepatocyte-specific expression.     

Human umbilical cord provides a significant source of unexpanded mesenchymal stromal cells

Background aimsHuman mesenchymal stromal cells (MSC) have considerable potential for cell-based therapies, including applications for regenerative medicine and immune suppression in graft-versus-host disease (GvHD). However, harvesting cells from the human body can cause iatrogenic disorders and in vitro expansion of MSC carries a risk of tumorigenesis and/or expansion of unexpected cell populationsMethodsGiven these problems, we have focused on umbilical cord, a tissue obtained with few ethical problems that contains significant numbers of MSC. We have developed a modified method to isolate MSC from umbilical cord, and investigated their properties using flow cytometry, mRNA analysis and an in vivo GvHD modelResultsOur study demonstrates that, using umbilical cord, large numbers of MSC can be safely obtained using a simple procedure without in vitro expansion, and these non-expanded MSC have the potential to suppress GvHD.ConclusionsOur results suggest that the combined banking of umbilical cord-derived MSC and identical cord blood-derived hematopoietic stem cell banking, where strict inspection of the infectious disease status of donors is performed, as well as further benefits of HLA-matched mesenchymal cells, could become one of the main sources of cells for cell-based therapy against various disorders.     

New approach to isolate mesenchymal stem cell (MSC) from human umbilical cord blood

HUCB (human umbilical cord blood) has been frequently used in clinical allogeneic HSC (haemopoietic stem cell) transplant. However, HUCB is poorly recognized as a rich source of MSC (mesenchymal stem cell). The aim of this study has been to establish a new method for isolating large number of MSC from HUCB to recognize it as a good source of MSC. HUCB samples were collected from women following their elective caesarean section. The new method (Clot Spot method) was carried out by explanting HUCB samples in mesencult complete medium and maintained in 37°C, in a 5% CO2 and air incubator. MSC presence was established by quantitative and qualitative immunophenotyping of cells and using FITC attached to MSC phenotypic markers (CD29, CD73, CD44 and CD105). Haematopoietic antibodies (CD34 and CD45) were used as negative control. MSC differentiation was examined in neurogenic and adipogenic media. Immunocytochemistry was carried out for the embryonic markers: SOX2 (sex determining region Y-box 2), OLIG-4 (oligodendrocyte-4) and FABP-4 (fatty acid binding protein-4). The new method was compared with the conventional Rosset Sep method. MSC cultures using the Clot Spot method showed 3-fold increase in proliferation rate compared with conventional method. Also, the cells showed high expression of MSC markers CD29, CD73, CD44 and CD105, but lacked the expression of specific HSC markers (CD34 and CD45). The isolated MSC showed some differentiation by expressing the neurogenic (SOX2 and Olig4) and adipogenic (FABP-4) markers respectively. In conclusion, HUCB is a good source of MSC using this new technique.     

Synergistic use of adult and embryonic stem cells to study human hematopoiesis

Embryonic stem cells (ESCs) and adult stem cells both provide important resources to define the mechanisms of hematopoietic cell development. To date, studies that utilize hematopoietic stem cells (HSCs) isolated from sites such as bone marrow or umbilical cord blood have been the primary means to identify molecular and phenotypic characteristics of blood cell populations able to mediate long-term hematopoietic engraftment. Although these HSCs are very useful clinically, they are difficult to expand in culture. Now, basic research on human ESCs provides opportunities for novel investigations into the mechanisms of HSC self-renewal. Eventually, the long history of basic and clinical research with adult hematopoietic cell transplantation could translate to establish human ESCs as a suitable alternative starting cell source for clinical hematopoietic reconstitution.     

Comparative intracellular cytokine production by in vitro stimulated T lymphocytes from human umbilical cord blood (HUCB) and adult peripheral blood (APB).

To date over 400 HUCB transplants have been reported from different centers. It has been suggested that there is a reduced graft-versus-host-disease (GVHD) with HUCB compared to bone marrow transplantation. Since cytokine production by a cell is an indication of the cells function it is important to determinate the differences between APB and HUCB with respect to production of these soluble factors. Our aim was to analyse the intracellular cytokine production by HUCB and APB T lymphocytes with and emphasize on their possible role in GVHD. Heparinized HUCB samples from 8 normal full-term deliveries and 10 normal blood donors were stimulated 4 hours at 37 degrees C and 5% CO2 with phorbol 12-myristate 13-acetate (PMA) and lonomycin in the presence of brefeldine. Afterwards cells were stained with CD3, CD4 or CD8 in different combinations. Finally, after cell permeabilization, cells were stained with Il-2, Il-4 or IFN-gamma. Data acquisition was performed on a FACScan flow cytometer. Compared to APB, HUCB T lymphocytes produced less Il-2, Il-4 and IFN-gamma. In HUCB, Il-2, Il-4 and IFN-gamma were produced predominantly by CD4+ T cells. In APB, Il-2 and Il-4 were also produced predominantly by CD4+ cells compared with CD8+ T lymphocytes, however, IFN-gamma was produced by both CD4+ and CD8+ T cells. These results indicate that there are clear differences in the cytokine profile between T cells in APB and HUCB.