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CD133− Cultivation in Neural Proliferation Media Differentiates Towards Neural Cell Lineages
Archives of Medical Research 42 (2011) 555e562
ORIGINAL ARTICLE
Umbilical Cord Blood Cells CD133þ/CD133 Cultivation in Neural Proliferation Media Differentiates Towards Neural Cell Lineages Lucia Slovinska,a Ivana Novotna,a Miroslav Kubes,b Jozef Radonak,c Stanislava Jergova,a Viera Cigankova,d Jan Rosocha,c and Dasa Cizkovaa a
Institute of Neurobiology, Center of Excellence, Slovak Academy of Sciences, Soltesovej, Kosice, Slovakia Slovak Placental Hematopoietic Stem Cell Registry, Eurocord, Slovakia, Dubravska, Bratislava, Slovakia c 1 Surgical Clinic, Faculty of Medicine, P.J. Safarik University and L. Pausteur Faculty Hospital, Trieda SNP1, Kosice, Slovakia d Institute of Histology and Embriology, The University of Veterinary Medicine and Pharmacy, Komenskeho, Kosice, Slovakia b
Received for publication April 7, 2011; accepted September 26, 2011 (ARCMED-D-11-00181).
Background and Aims. Umbilical cord blood (UCB) has been identified as a good source of hematopoietic and nonhematopoietic stem cells that can be easily isolated. In the present study we investigated the possibility of whether stem cells in mononuclear UCB grown under defined conditions can produce progeny with neural phenotype. Methods. A combination of antigen-driven magnetic cell sorting (MACs) method and defined culture conditions specific for cells of neural lineages were used for isolation, expansion and differentiation of CD133þ/ cells from UCB. Both UCB-derived fractions were expanded by exposure to growth factors (EGF, bFGF). Differentiation was induced by replacing them with fetal bovine serum. Using immunocytochemistry, the cell markers for neural (MAP2, GFAP, RIP) and non-neural lineages (S-100, von Willebrand factor) were detected. Results. The analysis revealed occurrence of fully mature neural and non-neural lineages, which showed qualitative and quantitative differences between population of CD133þ and CD133 cells. The expression levels of MAP2 and RIP in CD133þ were significantly higher than in CD133, more GFAP positive cells were found in the CD133. At the same time, S-100 was expressed by 32.47 6.24% of CD133 cells and 29.42 1.32% of CD133 cell expressed a von Willebrand factor antigen. Conclusions. Our results indicate that stem cells derived from umbilical cord blood are easy to obtain, proliferate and are able to differentiate towards the cells of neural lineages, which represents a promising way for their utilization in cell-based therapies for CNS injuries and diseases. Ó 2011 IMSS. Published by Elsevier Inc. Key Words: Umbilical cord blood, Magnetic cell sorting, CD133 cells, Neural lineage.
Introduction Umbilical cord blood (UCB) has been identified as a good source of hematopoietic stem cells that can be easily isolated, long-term cryopreserved and used afterwards to reconstitute the impaired blood lineages in various malignant and nonmalignant blood diseases (1). The first human cord blood application was accomplished in a patient with Fanconi’s anemia Address reprint requests to: Lucia Slovinska, Institute of Neurobiology, Center of Excellence, Slovak Academy of Sciences, Slotesovej 4-6, 04001, Kosice, Slovakia; Phone: þ421-55-678 5069; FAX: þ421-55-678 5074; E-mail: [email protected]
in 1988, which resulted in successful bone marrow reconstruction (2,3). This was an important finding as it proved UCB to be a powerful tool for treatment of several bloodrelated diseases such as leukemia (4), thalassemia and sickle cell disease (5). Recently, it has been demonstrated that umbilical cord blood-derived stem cells (UCB-SCs) have the potential to give rise also to nonhematopoietic cells such as bone, neural and endothelial cells (6). These studies concluded that the umbilical cord blood is most likely the mixture of different stem and progenitor cells such as blood, endothelial, and mesenchymal stem/progenitor cells. However, the main emphasis was focused on mesenchymal
0188-4409/$ - see front matter. Copyright Ó 2011 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2011.10.003
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stem cells that comprise a rare population of multipotent progenitors capable of both supporting hematopoiesis and differentiating into at least osteogenic, adipogenic and chondrogenic lineages (7,8). Some groups have reported the isolation of a mesenchymal progenitor cells from UCB hematopoietic progenitor cells by differential adherence selection (9e11), whereas others used lineage negative cell growth specific culture media (12). More surprisingly, exposing UCB cells to various experimental conditions showed that their progeny could also reveal properties typical for neuroectoderm-derived cells (13e17). This multilineage differentiation capacity and the expression of neural properties suggest that UCB cells may have the ability to transdifferentiate or become nonhematopoietic cells of various tissue lineages including neural cells (18), which may significantly contribute to a development of novel strategies for stem cell therapies of neurodegenerative disorders (19). In addition, it has been reported that these cells can be used as a source of therapeutically effective substances with the ability to improve functional outcomes after stroke (20e23), traumatic brain injury (24e26), spinal cord injury (27e30) and neurodegenerative disorders (19). Other transplantation studies have addressed the possibility whether the UCB mononuclear fraction contains nondifferentiated cells that may, under specific stimulatory circumstances such as stroke or placement into a favorable neurogenic environment (14), give rise to neural-like cells. In the present study we investigated whether stem cells in the mononuclear UCB fraction can produce progeny that express neural antigens when grown under conditions leading to progeny with neural phenotype. Using a combination of antigen-driven magnetic cell sorting method (magnetic separation of CD133þ cells) and appropriate culture conditions directing cells towards the neural lineage, we have analyzed the phenotypical characteristics of CD133þ/CD133 pools derived from UCB-SCs) that could be applied for cell-based therapies in central nervous system (CNS) diseases.
Materials and Methods Sample Collection Human umbilical cord blood samples (n 5 4) (70e90 mL/ unit) were obtained from healthy full-term pregnancies with parental informed consent of Eurocord-Slovakia. Only fresh cord blood processed within 12 h after birth was used in present study. Red blood cells were removed from the UBC by using Red Blood Cell Lysis buffer (RoboSep, STEMCELL Technologies, Vancouver, Canada) and the mononuclear cell fraction (MNC) was obtained by Ficoll density gradient method (Ficoll-Hypaque Plus, 1077 g/mL, Sigma, St. Louis, MO). Consecutively acquired UCB mononuclear cell fraction was processed for CD133 specific antibody-mediated magnetic cell sorting technology.
Cell Culture CD133 separation. CD133þ cells were isolated using a magnetic cell separation system, CD133 Isolation Kit (MACs, Miltenyi Biotec, Bergisch Gladbach, Germany), according to manufacturer’s instructions. After adding 100 ml FcR blocking reagent, cells were immediately incubated with CD133 antibody directly labeled with supermagnetic Micro Beads for 30 min at 4e8 C. Next, the cells were washed by adding 1e2 mL of separation buffer and centrifuged at 300 g for 10 min; the pellet was resuspended in 500 ml of separation buffer and processed to final, positive separation using MiniMACs Separator Column. Positively labeled CD133þ cells were flushed with 2 ml of separation buffer using a plunger fitted to column, centrifuged for 5 min at 300 g and washed with complete medium. Cell numbers and viability were determined in the hemocytometer by standard trypan blue method (0.4% trypan blue, Invitrogen, Carlsbad, CA). To assess the sorting, selected cells were stained by monoclonal antibody PE-conjugated CD133 (MACs, Miltenyi Biotec) and analyzed using cytospin Cytofuge-2 (StatSpin Cytofuge; Iris Sample Processing, Inc., Westwood, MA). The purity of CD133þ selected fraction was 91 4%. Cell culture proliferation and differentiation. Both UCBderived fractions (CD133þ and CD133 separated cells) were seeded on Nunc T25 culture flasks at a final concentration of 106 cells/mL in neural proliferation media (PM) containing Dulbecco’s modified Eagle Media (Gibco, Grand Island, NY) and Ham’s F12 (1/1 v/v) with 3 mM glucose, 5 mM HEPES (pH 7.2), supplemented with epidermal growth factor-EGF (20 ng/mL) (Gibco, Invitrogen), basic fibroblast growth factor-bFGF (20 ng/mL) (Gibco, Invitrogen), N2 supplement (10 ng/mL) (Gibco, Invitrogen), B27 supplement (10 ng/mL) (Gibco, Invitrogen), 1% penicillin-streptomycin (Biochrom AG, Berlin, DE) and incubated at 37 C, 5% CO2 conditions. The PM (half of volume) was changed every 3 days until the cells on the flask bottom reached O80% confluence (10e14 days). Afterwards, adherent cells were detached with 0.05% trypsin-EDTA (Gibco, Invitrogen) and replated into 24-well culture plates in growth factor-free differentiation media containing 10% fetal bovine serum (FBS). Cell cultures were grown for 12 days to induce differentiation and then fixed for immunocytochemical analysis. Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 20 min, washed with 0.1 M phosphatebuffered saline (PBS), blocked with 10% normal goat serum for 1 h at room temperature and then incubated overnight at 4 C with one of the following primary antibodies: mouse anti-GFAP (glial fibrillary acidic protein, 1:500, Millipore, Temecula, CA) for astrocytes, mouse anti-RIP (receptor interacting protein, 1:1000, Millipore, Temecula, CA)
Umbilical Cord Blood Cells in Neural Cultivation
for oligodendrocytes, rabbit anti-MAP2 (microtubule associated protein 2, 1:1000, Millipore, Temecula, CA) for neuronal cells, rabbit anti-S-100 (S-100 protein, 1:1000, DAKO, Carpinteria, CA) for glial cells, rabbit anti-von Willebrand factor (1:200, Millipore) for non-neural, endothelial cells. Afterwards, cell cultures were washed with 0.1 M PBS and incubated with species-appropriate fluorescent secondary antibodies (anti-mouse, anti-rabbit antibodies, Alexa Fluor 594, 488, 1:200, Molecular Probes, Eugene, OR) for 1 h at room temperature and by DAPI specific nuclear staining and viewed under the Nikon ECLIPSE Ti fluorescence microscope. Quantitative analysis. To determine the number of cells expressing neural or non-neural markers, the number of cells positive for each specific marker was counted as a percentage of total DAPI þ nuclei in 10 random visual fields of cells (400e600 cells per each UCB sample/per each marker). Data are presented as mean SEM. Statistical differences between groups were evaluated with Student’s t-test; level of significance was set at p !0.05.
Results In the present study we examined four umbilical cord blood samples obtained from healthy mothers at the time of birth and processed within 12 h after collection. The average volume of samples was 79.05 1.65 mL. These data correspond with parameters reported by Bieback et al. (31). Using magnetic cell separation method, a total of 6.7 x 105 CD133þ cells were isolated, which represented a rare population among mononuclear UCB cells (1.05 0.83%), with a viability maintained at 70e80% (Table 1). After MAC sorting, both isolated cell fractions, CD133þ and CD133, were maintained at the same optimized culture conditions (proliferation and differentiation media) enabling the generation of an adherent cell layer. The morphology of plated cells changed over time and cells reached 80e90% confluence by 10e14 days. The small, round cells did not form typical floating fraction, but more likely adhered to the plastic surface and created individual cell colonies composed of a few fibroblast-like cells. When subcultured into differentiation medium for 12 days, microscopic examination of cultures revealed the heterogeneous cell populations, which comprised a mixture of cells types ranging from large multipolar cells to spindle-shaped bipolar cells with fine, thin branching ends (Figure 1). Immunocytochemical analysis of separated fractions revealed occurrence of fully mature neural and non-neural lineages that showed qualitative and quantitative differences between populations of CD133þ and CD133 cells (Figure 2). Immunocytostaining of neural markers demonstrated that 39.45 3.95% of CD133þ cells expressed marker of differentiated neurons MAP2 (Figures 2 and 3),
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Table 1. Cord blood sample reults Cord blood Volume (mL) Nucleated cell (106 cells/mL) CD133þ (%) Viability (%)
(n 5 4) 79.05 64.10 1.05 73.50
1.65 13.10 0.83 2.22
which was statistically significantly higher ( p !0.05) in comparison with 11.78 3.48% of CD133 cells, respectively. Furthermore, we also detected positivity for mature glial antigens and for oligodendrocytes-RIP and astrocytes-GFAP (Figure 2). The number of differentiated glial cells was correlated in CD133þ vs. CD133 cell fractions: RIPþ oligodendrocytes 32.69 2.39% vs. 12.56 2.19%, GFAPþ astrocytes 18.97 3.16% vs. 24.28 8.79%. The statistically significant increase was observed in RIPþ oligodendrocytes, p !0.05 (Figure 3). The results are interesting because more GFAP positive cells were found in the population derived from CD133, although with no statistical significance. At the same time, S-100 was expressed by 32.47 6.24% of CD133 cells, and 29.42 1.32% of CD133 cell expressed a von Willebrand factor antigen (Figure 4). However, neither S-100 nor von Willebrand factor could be detected in the CD133þ population. There were no morphological differences between cells derived from both cellular fractions, although some variations in the expression of analyzed markers occurred. Thus, the fraction of CD133 cells generated not only neural, but also non-neural lineages regardless of the fact that the same cultivation condition was used for CD133þ cell fraction.
Discussion In the present study we examined the ability of MACs sorted CD133þ and CD133 UCB-SCs to produce progeny with neural properties that were directed by their maintenance in neural culture conditions. Bone marrow and UCB are the most frequently used stem cells for possible autologous transplantation. However, human UCB is probably the most attractive source of stem cells, which has turned out to be an excellent alternative also for clinical-scale allogeneic transplantation (32). Cord blood stem cells are easily accessible, immunologically naive and provide a source of stem cells without ethical dilemma. Furthermore, human UCB is used in the clinical treatment of O80 diseases most commonly related to immune and hematopoietic system disorders. Here we isolated UCB-SCs from fresh UCB samplesunits in accordance with the results of Bieback et al. (31). The average volume of UCB samples-units was 79.05 mL and the time between collection and isolation of cells was !12 h. Total nucleated and hematopoietic stem cell content of UCB is significantly affected by the baby’s birth weight,
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Figure 1. Phase contrast images of human umbilical cord blood CD133þ separated cells that were able to generate an adherent monolayer. The morphology of plated cells changed over time. These initially small, round cells (A) acquired a new morphological appearance within cultivation. Microscopic examination of cultures revealed a heterogeneous mixture of cell (B,F) types ranging from large cells (C), fibroblast-like cells (D) to spindle-shaped cells with fine, thin branching ends (E). Scale bar 5 100 mm. DIV, days in vitro.
mother’s age at delivery, mother’s obstetric history, and gestational stage (33). Therefore, in the present experiment we have used all four UBC units that could not be processed for further cryopreservation and storage due to low content of nucleated cell within accomplished volumes and would be otherwise discarded. It is important to point out that in addition to freshly harvested UCB samples, cryopreserved human UCB fractions may also be used as an alternative source of SCs for experimental use and eventually for clinical application. The issues of cryopreservation of UBC-units (recovery, viability and stability of phenotype lineage features after thawing) are the most critical factors that should be considered in relation to their potential autologous/allogeneic clinical applications. However, during the last few years the isolation, characterization, and separation as well as expansion of UCB-SCs was
performed from fresh as well as from cryopreserved UCB units. For example, Lee et al. (7) obtained homogeneous mesenchymal stem cells from the MNC fraction of cryopreserved UCB by plastic adherence procedure. These cells exhibited fibroblast-like morphology and typical mesenchymal phenotype, expressing CD73, CD105 and CD166. Other groups developed a negative immunomagnetic selection method that depletes hematopoietic lineage markerexpressing cells from fresh human UCB (hUCB), thereby isolating a discrete lineage-negative stem cell population (0.1% of mononuclear hUCB). These hematopoietic hUCB cells expanded into primitive nonadherent progenitors and simultaneously produced slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks (34). According to several known separation methods used for isolation of various progenitor cells (9,11), an antigen-driven
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Figure 2. Cell-type-specific immunostaining of CD133þ cells: for MAP2 neurons (A), GFAP astrocytes (B), RIP oligodendrocytes (C), and CD133 cell fraction: for neurons (A), astrocytes (B), oligodendrocytes (C); differentiated in FBS enriched, growth factor-free medium. No morphological differences were observed between cells staining for each marker derived from both cell fractions. Scale bar 5 100 mm.
magnetic cell sorting method (anti-CD133 antigen-specific antibody based magnetic separation) was used in our study. This method enabled us to isolate CD133þ and CD133 cells from UBC. CD133 positive cells represent self-renewing noncommitted stem/progenitor cells that have the potential to differentiate into myelomonocytic lineage (35), endothelial cells (36), and cardiomyocytes (37) as well as to neural cells in the presence of different lineage inducers. Additionally, in CD133þ cells due to upregulation of genes involved in angiogenesis, permeability may support tumor growth and could be a target biomarker for anticancer therapy (38,39). Although CD133 is suggested to be a marker of primitive hematopoietic stem/progenitor cells, cellular mechanisms involved in the regulation of CD133 and their function remain unknown (40). In the present experimental study, both fractions (CD133þ and CD133) were
cultivated under the same conditions in standardized neural defined medium required for optimal neural progenitor expansion and final differentiation (41). This was in accordance with data showing that the fate of UCB-SCs could be directed toward desirable cell lineage when using the appropriate culture condition media. For example, it has been documented that when exposed to medium supporting neural cell differentiation and growth, UCB-derived multipotential stem cells give rise to neurons, astrocytes, oligodendrocytes and Schwann cells (12). Particularly, when UCB-SCs are exposed to hEGF/bFGF for 1 week, their morphological features appeared to acquire some of the attributes of neural cells in culture such as long bipolar extension and branching ends, while expressing neuralspecific antigens (6). There are several studies confirming that UCB-derived mononuclear cells can differentiate into various types of cells (42) including neurons and glial cells
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Figure 3. Immunophenotype analysis of UCB-SCs CD133 separated cells. Comparison of percentage of immunopositive cells derived from CD133þ (white bars) and CD133 (black bars) fraction of UCB-SCs cells. The CD133 magnetic separation of UCB-SC and proliferation and differentiation of isolated CD133þ/ fractions resulted in different cell immunophenotypes: a statistically significant increase was observed in neurons MAP2þ level and oligodendrocytes RIPþ level. A decrease was observed in astrocytes GFAPþ level of CD133þ. In the CD133 fraction, cells with non-neural phenotypes were also detected. Data are presented as mean SEM (*p !0.05).
by exposing them to various inducers such as retinoic acid (RA) (43), RA in combination with nerve growth factor (NGF) and brain-derived growth factor (BDNF) (44), b-mercaptoethanol (45), and EGF as well as bFGF (36,46). In the present study, isolated CD133þ and CD133 cells were expanded by exposure to growth factors (hEGF, hbFGF), whereas the differentiation was induced by replacing them with fetal bovine serum. The morphology of plated cells changed over time and was comparable in both cell fractions, ranging from large flat cells to spindle-shaped cells with fine processes, mimicking neuronal features. This was confirmed by the incidence of all three main neural antigens, for neuronseMAP2, astrocyteseGFAP and oligodendrocyteseRIP that were expressed in both CD133þ and CD133 cell fractions, but in different
quantities. A significantly higher number of MAP2þ and RIPþ cells occurred in CD133þ population, whereas increased numbers of GFAP-positive astrocytes were found in fractions derived from CD133. At the same time, only CD133 derived cells expressed von Willebrand factor, an antigen typical for endothelial cells and S-100 protein, which is involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation and was found in glial and ependymal cells (47,48). Although it has been shown that various inducers, specifically defined media or separation methods may direct UCB stem cells towards neural lineages, it is still not clear whether they may represent functional neurons or glial cells. Some results demonstrate that neuronal cells are generated from CD133þ/CD34 progenitors in vitro, but also that they require co-culture with CD133 cells to undergo neuronal differentiation (49). However, preclinical transplantation studies have shown that UCB-SCs are capable to engraft and survive within injured brain areas, resulting in reduced post-stroke behavioral deficits in rats (21). The possible beneficial efficacy of delivered UCB-SCs may be through inflammatory modulation and stimulation of angiogenesis and neurogenesis which, together, most likely contribute to the functional recovery in the animal model of stroke (26,50,51). It is well established that angiogenesis is an important healing process of wounds including tissue ischemia, which appears to promote neurogenesis in neurological disease. We also confirmed the presence of endothelial progenitor cells (EPCs) in the CD133 fraction, which give rise to mature endothelial cells expressing von Willebrand factor that may contribute to the revascularization of damaged tissue. In conclusion, these results indicate that UBC-SCs are relatively easy to obtain and are able to proliferate and differentiate towards the cells of neural lineages. MACs sorting enabled us to gain CD133þ population with a significantly higher content of neurons and oligodendroglial cells
Figure 4. Cell-type-specific immunostaining of CD133 cells for S-100 glial cells (A) and von Willebrand factor endothelial cells (B) differentiated in FBS enriched, growth factor-free medium. Scale bar 5 100 mm. The S-100 glial and von Willebrand factor markers were not found in the CD133þ cell fraction.
Umbilical Cord Blood Cells in Neural Cultivation
and decreased amount of astrocytes when compared to CD133 population. Therefore, the cellular composition of CD133þ fraction and their proportional share may represent a valuable neurogenic source for cell-based therapies of various CNS injuries and diseases. Also, the CD133 fraction with a reduced content of neural and non-neural pools may be considered as an important stem cell source for ongoing clinical research and trials. Acknowledgments This work was supported by grant project VEGA 1-0674-09, 2/ 0114/11, MVTS COST BM 1002.
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ORIGINAL ARTICLE
Umbilical Cord Blood Cells CD133þ/CD133 Cultivation in Neural Proliferation Media Differentiates Towards Neural Cell Lineages Lucia Slovinska,a Ivana Novotna,a Miroslav Kubes,b Jozef Radonak,c Stanislava Jergova,a Viera Cigankova,d Jan Rosocha,c and Dasa Cizkovaa a
Institute of Neurobiology, Center of Excellence, Slovak Academy of Sciences, Soltesovej, Kosice, Slovakia Slovak Placental Hematopoietic Stem Cell Registry, Eurocord, Slovakia, Dubravska, Bratislava, Slovakia c 1 Surgical Clinic, Faculty of Medicine, P.J. Safarik University and L. Pausteur Faculty Hospital, Trieda SNP1, Kosice, Slovakia d Institute of Histology and Embriology, The University of Veterinary Medicine and Pharmacy, Komenskeho, Kosice, Slovakia b
Received for publication April 7, 2011; accepted September 26, 2011 (ARCMED-D-11-00181).
Background and Aims. Umbilical cord blood (UCB) has been identified as a good source of hematopoietic and nonhematopoietic stem cells that can be easily isolated. In the present study we investigated the possibility of whether stem cells in mononuclear UCB grown under defined conditions can produce progeny with neural phenotype. Methods. A combination of antigen-driven magnetic cell sorting (MACs) method and defined culture conditions specific for cells of neural lineages were used for isolation, expansion and differentiation of CD133þ/ cells from UCB. Both UCB-derived fractions were expanded by exposure to growth factors (EGF, bFGF). Differentiation was induced by replacing them with fetal bovine serum. Using immunocytochemistry, the cell markers for neural (MAP2, GFAP, RIP) and non-neural lineages (S-100, von Willebrand factor) were detected. Results. The analysis revealed occurrence of fully mature neural and non-neural lineages, which showed qualitative and quantitative differences between population of CD133þ and CD133 cells. The expression levels of MAP2 and RIP in CD133þ were significantly higher than in CD133, more GFAP positive cells were found in the CD133. At the same time, S-100 was expressed by 32.47 6.24% of CD133 cells and 29.42 1.32% of CD133 cell expressed a von Willebrand factor antigen. Conclusions. Our results indicate that stem cells derived from umbilical cord blood are easy to obtain, proliferate and are able to differentiate towards the cells of neural lineages, which represents a promising way for their utilization in cell-based therapies for CNS injuries and diseases. Ó 2011 IMSS. Published by Elsevier Inc. Key Words: Umbilical cord blood, Magnetic cell sorting, CD133 cells, Neural lineage.
Introduction Umbilical cord blood (UCB) has been identified as a good source of hematopoietic stem cells that can be easily isolated, long-term cryopreserved and used afterwards to reconstitute the impaired blood lineages in various malignant and nonmalignant blood diseases (1). The first human cord blood application was accomplished in a patient with Fanconi’s anemia Address reprint requests to: Lucia Slovinska, Institute of Neurobiology, Center of Excellence, Slovak Academy of Sciences, Slotesovej 4-6, 04001, Kosice, Slovakia; Phone: þ421-55-678 5069; FAX: þ421-55-678 5074; E-mail: [email protected]
in 1988, which resulted in successful bone marrow reconstruction (2,3). This was an important finding as it proved UCB to be a powerful tool for treatment of several bloodrelated diseases such as leukemia (4), thalassemia and sickle cell disease (5). Recently, it has been demonstrated that umbilical cord blood-derived stem cells (UCB-SCs) have the potential to give rise also to nonhematopoietic cells such as bone, neural and endothelial cells (6). These studies concluded that the umbilical cord blood is most likely the mixture of different stem and progenitor cells such as blood, endothelial, and mesenchymal stem/progenitor cells. However, the main emphasis was focused on mesenchymal
0188-4409/$ - see front matter. Copyright Ó 2011 IMSS. Published by Elsevier Inc. doi: 10.1016/j.arcmed.2011.10.003
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stem cells that comprise a rare population of multipotent progenitors capable of both supporting hematopoiesis and differentiating into at least osteogenic, adipogenic and chondrogenic lineages (7,8). Some groups have reported the isolation of a mesenchymal progenitor cells from UCB hematopoietic progenitor cells by differential adherence selection (9e11), whereas others used lineage negative cell growth specific culture media (12). More surprisingly, exposing UCB cells to various experimental conditions showed that their progeny could also reveal properties typical for neuroectoderm-derived cells (13e17). This multilineage differentiation capacity and the expression of neural properties suggest that UCB cells may have the ability to transdifferentiate or become nonhematopoietic cells of various tissue lineages including neural cells (18), which may significantly contribute to a development of novel strategies for stem cell therapies of neurodegenerative disorders (19). In addition, it has been reported that these cells can be used as a source of therapeutically effective substances with the ability to improve functional outcomes after stroke (20e23), traumatic brain injury (24e26), spinal cord injury (27e30) and neurodegenerative disorders (19). Other transplantation studies have addressed the possibility whether the UCB mononuclear fraction contains nondifferentiated cells that may, under specific stimulatory circumstances such as stroke or placement into a favorable neurogenic environment (14), give rise to neural-like cells. In the present study we investigated whether stem cells in the mononuclear UCB fraction can produce progeny that express neural antigens when grown under conditions leading to progeny with neural phenotype. Using a combination of antigen-driven magnetic cell sorting method (magnetic separation of CD133þ cells) and appropriate culture conditions directing cells towards the neural lineage, we have analyzed the phenotypical characteristics of CD133þ/CD133 pools derived from UCB-SCs) that could be applied for cell-based therapies in central nervous system (CNS) diseases.
Materials and Methods Sample Collection Human umbilical cord blood samples (n 5 4) (70e90 mL/ unit) were obtained from healthy full-term pregnancies with parental informed consent of Eurocord-Slovakia. Only fresh cord blood processed within 12 h after birth was used in present study. Red blood cells were removed from the UBC by using Red Blood Cell Lysis buffer (RoboSep, STEMCELL Technologies, Vancouver, Canada) and the mononuclear cell fraction (MNC) was obtained by Ficoll density gradient method (Ficoll-Hypaque Plus, 1077 g/mL, Sigma, St. Louis, MO). Consecutively acquired UCB mononuclear cell fraction was processed for CD133 specific antibody-mediated magnetic cell sorting technology.
Cell Culture CD133 separation. CD133þ cells were isolated using a magnetic cell separation system, CD133 Isolation Kit (MACs, Miltenyi Biotec, Bergisch Gladbach, Germany), according to manufacturer’s instructions. After adding 100 ml FcR blocking reagent, cells were immediately incubated with CD133 antibody directly labeled with supermagnetic Micro Beads for 30 min at 4e8 C. Next, the cells were washed by adding 1e2 mL of separation buffer and centrifuged at 300 g for 10 min; the pellet was resuspended in 500 ml of separation buffer and processed to final, positive separation using MiniMACs Separator Column. Positively labeled CD133þ cells were flushed with 2 ml of separation buffer using a plunger fitted to column, centrifuged for 5 min at 300 g and washed with complete medium. Cell numbers and viability were determined in the hemocytometer by standard trypan blue method (0.4% trypan blue, Invitrogen, Carlsbad, CA). To assess the sorting, selected cells were stained by monoclonal antibody PE-conjugated CD133 (MACs, Miltenyi Biotec) and analyzed using cytospin Cytofuge-2 (StatSpin Cytofuge; Iris Sample Processing, Inc., Westwood, MA). The purity of CD133þ selected fraction was 91 4%. Cell culture proliferation and differentiation. Both UCBderived fractions (CD133þ and CD133 separated cells) were seeded on Nunc T25 culture flasks at a final concentration of 106 cells/mL in neural proliferation media (PM) containing Dulbecco’s modified Eagle Media (Gibco, Grand Island, NY) and Ham’s F12 (1/1 v/v) with 3 mM glucose, 5 mM HEPES (pH 7.2), supplemented with epidermal growth factor-EGF (20 ng/mL) (Gibco, Invitrogen), basic fibroblast growth factor-bFGF (20 ng/mL) (Gibco, Invitrogen), N2 supplement (10 ng/mL) (Gibco, Invitrogen), B27 supplement (10 ng/mL) (Gibco, Invitrogen), 1% penicillin-streptomycin (Biochrom AG, Berlin, DE) and incubated at 37 C, 5% CO2 conditions. The PM (half of volume) was changed every 3 days until the cells on the flask bottom reached O80% confluence (10e14 days). Afterwards, adherent cells were detached with 0.05% trypsin-EDTA (Gibco, Invitrogen) and replated into 24-well culture plates in growth factor-free differentiation media containing 10% fetal bovine serum (FBS). Cell cultures were grown for 12 days to induce differentiation and then fixed for immunocytochemical analysis. Immunocytochemistry. Cells were fixed with 4% paraformaldehyde for 20 min, washed with 0.1 M phosphatebuffered saline (PBS), blocked with 10% normal goat serum for 1 h at room temperature and then incubated overnight at 4 C with one of the following primary antibodies: mouse anti-GFAP (glial fibrillary acidic protein, 1:500, Millipore, Temecula, CA) for astrocytes, mouse anti-RIP (receptor interacting protein, 1:1000, Millipore, Temecula, CA)
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for oligodendrocytes, rabbit anti-MAP2 (microtubule associated protein 2, 1:1000, Millipore, Temecula, CA) for neuronal cells, rabbit anti-S-100 (S-100 protein, 1:1000, DAKO, Carpinteria, CA) for glial cells, rabbit anti-von Willebrand factor (1:200, Millipore) for non-neural, endothelial cells. Afterwards, cell cultures were washed with 0.1 M PBS and incubated with species-appropriate fluorescent secondary antibodies (anti-mouse, anti-rabbit antibodies, Alexa Fluor 594, 488, 1:200, Molecular Probes, Eugene, OR) for 1 h at room temperature and by DAPI specific nuclear staining and viewed under the Nikon ECLIPSE Ti fluorescence microscope. Quantitative analysis. To determine the number of cells expressing neural or non-neural markers, the number of cells positive for each specific marker was counted as a percentage of total DAPI þ nuclei in 10 random visual fields of cells (400e600 cells per each UCB sample/per each marker). Data are presented as mean SEM. Statistical differences between groups were evaluated with Student’s t-test; level of significance was set at p !0.05.
Results In the present study we examined four umbilical cord blood samples obtained from healthy mothers at the time of birth and processed within 12 h after collection. The average volume of samples was 79.05 1.65 mL. These data correspond with parameters reported by Bieback et al. (31). Using magnetic cell separation method, a total of 6.7 x 105 CD133þ cells were isolated, which represented a rare population among mononuclear UCB cells (1.05 0.83%), with a viability maintained at 70e80% (Table 1). After MAC sorting, both isolated cell fractions, CD133þ and CD133, were maintained at the same optimized culture conditions (proliferation and differentiation media) enabling the generation of an adherent cell layer. The morphology of plated cells changed over time and cells reached 80e90% confluence by 10e14 days. The small, round cells did not form typical floating fraction, but more likely adhered to the plastic surface and created individual cell colonies composed of a few fibroblast-like cells. When subcultured into differentiation medium for 12 days, microscopic examination of cultures revealed the heterogeneous cell populations, which comprised a mixture of cells types ranging from large multipolar cells to spindle-shaped bipolar cells with fine, thin branching ends (Figure 1). Immunocytochemical analysis of separated fractions revealed occurrence of fully mature neural and non-neural lineages that showed qualitative and quantitative differences between populations of CD133þ and CD133 cells (Figure 2). Immunocytostaining of neural markers demonstrated that 39.45 3.95% of CD133þ cells expressed marker of differentiated neurons MAP2 (Figures 2 and 3),
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Table 1. Cord blood sample reults Cord blood Volume (mL) Nucleated cell (106 cells/mL) CD133þ (%) Viability (%)
(n 5 4) 79.05 64.10 1.05 73.50
1.65 13.10 0.83 2.22
which was statistically significantly higher ( p !0.05) in comparison with 11.78 3.48% of CD133 cells, respectively. Furthermore, we also detected positivity for mature glial antigens and for oligodendrocytes-RIP and astrocytes-GFAP (Figure 2). The number of differentiated glial cells was correlated in CD133þ vs. CD133 cell fractions: RIPþ oligodendrocytes 32.69 2.39% vs. 12.56 2.19%, GFAPþ astrocytes 18.97 3.16% vs. 24.28 8.79%. The statistically significant increase was observed in RIPþ oligodendrocytes, p !0.05 (Figure 3). The results are interesting because more GFAP positive cells were found in the population derived from CD133, although with no statistical significance. At the same time, S-100 was expressed by 32.47 6.24% of CD133 cells, and 29.42 1.32% of CD133 cell expressed a von Willebrand factor antigen (Figure 4). However, neither S-100 nor von Willebrand factor could be detected in the CD133þ population. There were no morphological differences between cells derived from both cellular fractions, although some variations in the expression of analyzed markers occurred. Thus, the fraction of CD133 cells generated not only neural, but also non-neural lineages regardless of the fact that the same cultivation condition was used for CD133þ cell fraction.
Discussion In the present study we examined the ability of MACs sorted CD133þ and CD133 UCB-SCs to produce progeny with neural properties that were directed by their maintenance in neural culture conditions. Bone marrow and UCB are the most frequently used stem cells for possible autologous transplantation. However, human UCB is probably the most attractive source of stem cells, which has turned out to be an excellent alternative also for clinical-scale allogeneic transplantation (32). Cord blood stem cells are easily accessible, immunologically naive and provide a source of stem cells without ethical dilemma. Furthermore, human UCB is used in the clinical treatment of O80 diseases most commonly related to immune and hematopoietic system disorders. Here we isolated UCB-SCs from fresh UCB samplesunits in accordance with the results of Bieback et al. (31). The average volume of UCB samples-units was 79.05 mL and the time between collection and isolation of cells was !12 h. Total nucleated and hematopoietic stem cell content of UCB is significantly affected by the baby’s birth weight,
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Figure 1. Phase contrast images of human umbilical cord blood CD133þ separated cells that were able to generate an adherent monolayer. The morphology of plated cells changed over time. These initially small, round cells (A) acquired a new morphological appearance within cultivation. Microscopic examination of cultures revealed a heterogeneous mixture of cell (B,F) types ranging from large cells (C), fibroblast-like cells (D) to spindle-shaped cells with fine, thin branching ends (E). Scale bar 5 100 mm. DIV, days in vitro.
mother’s age at delivery, mother’s obstetric history, and gestational stage (33). Therefore, in the present experiment we have used all four UBC units that could not be processed for further cryopreservation and storage due to low content of nucleated cell within accomplished volumes and would be otherwise discarded. It is important to point out that in addition to freshly harvested UCB samples, cryopreserved human UCB fractions may also be used as an alternative source of SCs for experimental use and eventually for clinical application. The issues of cryopreservation of UBC-units (recovery, viability and stability of phenotype lineage features after thawing) are the most critical factors that should be considered in relation to their potential autologous/allogeneic clinical applications. However, during the last few years the isolation, characterization, and separation as well as expansion of UCB-SCs was
performed from fresh as well as from cryopreserved UCB units. For example, Lee et al. (7) obtained homogeneous mesenchymal stem cells from the MNC fraction of cryopreserved UCB by plastic adherence procedure. These cells exhibited fibroblast-like morphology and typical mesenchymal phenotype, expressing CD73, CD105 and CD166. Other groups developed a negative immunomagnetic selection method that depletes hematopoietic lineage markerexpressing cells from fresh human UCB (hUCB), thereby isolating a discrete lineage-negative stem cell population (0.1% of mononuclear hUCB). These hematopoietic hUCB cells expanded into primitive nonadherent progenitors and simultaneously produced slow-dividing adherent cells with neuroglial progenitor cell morphology over 8 weeks (34). According to several known separation methods used for isolation of various progenitor cells (9,11), an antigen-driven
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Figure 2. Cell-type-specific immunostaining of CD133þ cells: for MAP2 neurons (A), GFAP astrocytes (B), RIP oligodendrocytes (C), and CD133 cell fraction: for neurons (A), astrocytes (B), oligodendrocytes (C); differentiated in FBS enriched, growth factor-free medium. No morphological differences were observed between cells staining for each marker derived from both cell fractions. Scale bar 5 100 mm.
magnetic cell sorting method (anti-CD133 antigen-specific antibody based magnetic separation) was used in our study. This method enabled us to isolate CD133þ and CD133 cells from UBC. CD133 positive cells represent self-renewing noncommitted stem/progenitor cells that have the potential to differentiate into myelomonocytic lineage (35), endothelial cells (36), and cardiomyocytes (37) as well as to neural cells in the presence of different lineage inducers. Additionally, in CD133þ cells due to upregulation of genes involved in angiogenesis, permeability may support tumor growth and could be a target biomarker for anticancer therapy (38,39). Although CD133 is suggested to be a marker of primitive hematopoietic stem/progenitor cells, cellular mechanisms involved in the regulation of CD133 and their function remain unknown (40). In the present experimental study, both fractions (CD133þ and CD133) were
cultivated under the same conditions in standardized neural defined medium required for optimal neural progenitor expansion and final differentiation (41). This was in accordance with data showing that the fate of UCB-SCs could be directed toward desirable cell lineage when using the appropriate culture condition media. For example, it has been documented that when exposed to medium supporting neural cell differentiation and growth, UCB-derived multipotential stem cells give rise to neurons, astrocytes, oligodendrocytes and Schwann cells (12). Particularly, when UCB-SCs are exposed to hEGF/bFGF for 1 week, their morphological features appeared to acquire some of the attributes of neural cells in culture such as long bipolar extension and branching ends, while expressing neuralspecific antigens (6). There are several studies confirming that UCB-derived mononuclear cells can differentiate into various types of cells (42) including neurons and glial cells
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Figure 3. Immunophenotype analysis of UCB-SCs CD133 separated cells. Comparison of percentage of immunopositive cells derived from CD133þ (white bars) and CD133 (black bars) fraction of UCB-SCs cells. The CD133 magnetic separation of UCB-SC and proliferation and differentiation of isolated CD133þ/ fractions resulted in different cell immunophenotypes: a statistically significant increase was observed in neurons MAP2þ level and oligodendrocytes RIPþ level. A decrease was observed in astrocytes GFAPþ level of CD133þ. In the CD133 fraction, cells with non-neural phenotypes were also detected. Data are presented as mean SEM (*p !0.05).
by exposing them to various inducers such as retinoic acid (RA) (43), RA in combination with nerve growth factor (NGF) and brain-derived growth factor (BDNF) (44), b-mercaptoethanol (45), and EGF as well as bFGF (36,46). In the present study, isolated CD133þ and CD133 cells were expanded by exposure to growth factors (hEGF, hbFGF), whereas the differentiation was induced by replacing them with fetal bovine serum. The morphology of plated cells changed over time and was comparable in both cell fractions, ranging from large flat cells to spindle-shaped cells with fine processes, mimicking neuronal features. This was confirmed by the incidence of all three main neural antigens, for neuronseMAP2, astrocyteseGFAP and oligodendrocyteseRIP that were expressed in both CD133þ and CD133 cell fractions, but in different
quantities. A significantly higher number of MAP2þ and RIPþ cells occurred in CD133þ population, whereas increased numbers of GFAP-positive astrocytes were found in fractions derived from CD133. At the same time, only CD133 derived cells expressed von Willebrand factor, an antigen typical for endothelial cells and S-100 protein, which is involved in the regulation of a number of cellular processes such as cell cycle progression and differentiation and was found in glial and ependymal cells (47,48). Although it has been shown that various inducers, specifically defined media or separation methods may direct UCB stem cells towards neural lineages, it is still not clear whether they may represent functional neurons or glial cells. Some results demonstrate that neuronal cells are generated from CD133þ/CD34 progenitors in vitro, but also that they require co-culture with CD133 cells to undergo neuronal differentiation (49). However, preclinical transplantation studies have shown that UCB-SCs are capable to engraft and survive within injured brain areas, resulting in reduced post-stroke behavioral deficits in rats (21). The possible beneficial efficacy of delivered UCB-SCs may be through inflammatory modulation and stimulation of angiogenesis and neurogenesis which, together, most likely contribute to the functional recovery in the animal model of stroke (26,50,51). It is well established that angiogenesis is an important healing process of wounds including tissue ischemia, which appears to promote neurogenesis in neurological disease. We also confirmed the presence of endothelial progenitor cells (EPCs) in the CD133 fraction, which give rise to mature endothelial cells expressing von Willebrand factor that may contribute to the revascularization of damaged tissue. In conclusion, these results indicate that UBC-SCs are relatively easy to obtain and are able to proliferate and differentiate towards the cells of neural lineages. MACs sorting enabled us to gain CD133þ population with a significantly higher content of neurons and oligodendroglial cells
Figure 4. Cell-type-specific immunostaining of CD133 cells for S-100 glial cells (A) and von Willebrand factor endothelial cells (B) differentiated in FBS enriched, growth factor-free medium. Scale bar 5 100 mm. The S-100 glial and von Willebrand factor markers were not found in the CD133þ cell fraction.
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and decreased amount of astrocytes when compared to CD133 population. Therefore, the cellular composition of CD133þ fraction and their proportional share may represent a valuable neurogenic source for cell-based therapies of various CNS injuries and diseases. Also, the CD133 fraction with a reduced content of neural and non-neural pools may be considered as an important stem cell source for ongoing clinical research and trials. Acknowledgments This work was supported by grant project VEGA 1-0674-09, 2/ 0114/11, MVTS COST BM 1002.
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