Human cord blood-derived cells attain neuronal and glial features in vitro

Long-standing dogma states that neural stem cells with the ability to differentiate into neurons, astrocytes and oligodendrocytes are derived embryologically from neuroepithelial progenitors. However, several studies from different laboratories have recently reported that human or rodent mesenchymal bone marrow (BM) cells can be directed in vivo and in vitro into a neuronal or astrocytic fate (Azizi et al.,1998; Kopen et al.,1999; Sanchez-Ramos et al.,2000; Woodbury et al.,2000). Vice versa, neural stem cells isolated from the adult brain can develop blood cell elements (Bjornson et al., 1999) and give rise to all germ cell layers(Clarke et al., 2000) owing to the surprising versatility of their differentiation program. Moreover, rodent BM cells can invade the brain of previously irradiated or genetically myeloid-ablated recipients and give rise not only to mesenchyma-derived brain macrophages or microglia but also to astroglia and neurons, which were previously thought to belong exclusively to the neuroepithelial lineage(Eglitis and Mezey, 1997;Brazelton et al., 2000;Mezey et al., 2000). The above data together with the recently reported ability of stem cells residing in various organs to omit tissue-restricted specification(Alison et al., 2000;Krause et al., 2001;Zuk et al., 2001) suggest that classical barriers of cell differentiation can be broken down under certain permissive conditions, rendering the dogmatic `point of no return' of cellular lineage inaccurate. These results also indicated a remarkable plasticity in tissue-specific stem cells and encouraged us to look for a source of precursors that could be committed to neural fate in tissues other than neuroectodermal.

We selected umbilical cord blood (CB) cells for our study. These cells are easily available and preserved, and they could potentially serve as a routine starting material for isolation and expansion of cells for allogenic as well as authologous transplantations. The preliminary results of this study have already been presented(Bużańska et al.,2001a; Machaj et al.,2001). Here, using the method of CB cell subfractionation and their subsequent culturing in the presence of defined media and growth factors, we were able to generate a self-renewing, clonogenic cell population with neural-type precursor characteristics.

Cord blood was collected after obtaining the approval of a local Ethical Committee and the mother's informed consent. Blood was collected after delivery of the placenta by puncturing umbilical cord veins (>40 ml/sample). Cell numbers, viability and blood sterility were evaluated, and blood storage time did not exceed 12 hours. The mononuclear cell fraction was isolated on a Ficoll/Hypaque gradient, and the cells that bound to the immunomagnetic beads coated with anti-CD34 antibody were eliminated by immunomagnetic sorting (MilteneyiBiotek anti-CD34 Isolation Kit). After washing, the remaining cells were resuspended in Iscove's modified Dulbecco's(IMDM, Gibco) medium supplemented with 10% fetal calf serum (FCS, Gibco) at a final concentration of 10⁶ cells/ml. Plastic-adherent cells were cultured for 3 weeks in IMDM plus 10% FCS at 37°C, 5% CO₂, in a fully humidified atmosphere and with 50% of the media being changed every week. Before reaching the monolayer phase, cells were trypsimised and re-cultured in similar conditions for the next 3 weeks. At the beginning and the end of the culturing period, the cells were analysed by flow cytometry.

A Becton-Dickinson FACS Scan and commercial antibodies (HPCA-2 anti-CD34 phycoerithrine-conjugated and anti-CD45 fluoresceine-conjugated) were used for FACS examination.

For cell culture expansion, trypsin-removed cells were plated in plastic 25 cm² culture flasks at a density of 5×10⁴cells/cm² in DMEM (Gibco) supplemented with 10% FCS, EGF (epidermal growth factor, Sigma) at 10 ng/ml and antibiotic-antimycotic solution (AAS,Sigma, 1:100). The cells were grown for 7 days to obtain a monolayer. Some confluent cultures were re-seeded after trypsinisation, whereas some were kept for 5 days longer in order to obtain free floating, non-adherent cells. Both kinds of cell, when transferred to separate flasks or multi-well plates at a density of about 10 cells/cm² in the presence of EGF, started to grow clones within the next 7 days in culture. The clones were observed to grown in size during the following 14 days. As the cells proliferated, some of them detached from the plastic and remained floating in suspension; however,they stayed viable and could give rise to new clones. After reseeding, these cells can be maintained as an adherent, undifferentiated, clonogenic population in the presence of EGF and FCS during the six, already tested,passages.

In additional experiments, these cells were cultured in the commercially available clonal cell culture system (Methocult H 4330, Stem Cell Technologies), which support the growth of both erythroid and myeloid precursors. The cells were analysed for the possible appearance of erythropoietic (BFU-E), granulo/macrophagopoietic (GM-CFC) and mixed(CFU-GEMM) colonies after 14 days of culture at 37°C in 5% CO₂in a fully humidified atmosphere.

Clones that had been grown for 14 days in the conditions described above were treated directly with Neurobasal Media supplemented with 10% FCS and 0.5μM all-trans-retinoic acid (RA, Sigma) for the following 4 days.

In separate experiments, clone-growing cells were collected by trypsinisation and plated on poly-L-lysine 24-well tissue culture plates at a density 5×10⁴ cells/cm². The media used for promoting cellular differentiation was as follow: (1) Neurobasal Medium(Gibco) supplemented with 10% FCS (Gibco); (2) Neurobasal Medium supplemented with 10% FCS and 0.5 μM RA; (3) Neurobasal Medium supplemented with 10%FCS, 0.5 μM RA and BDNF (Sigma) at a concentration of 10 ng/ml.

In each case, cells were incubated at 37°C in 5% CO₂ in a fully humidified atmosphere for 4 days and fixed for immunocytochemical detection of neural-specific antigens.

Mixed primary cultures were prepared from the brain cortex of 18-19 day-old rat embryos (Wistar) under sterile conditions. Dissected tissue was placed in Ca²⁺- and Mg²⁺-free HBSS (Gibco), dispersed mechanically(10-12 pipette strokes) and then enzymatically by a 15 minute incubation in 0.2% trypsin (Gibco). After centrifugation at 200 g for 3 minutes the pellets were resuspended in Dulbecco's modified Eagle's medium(DMEM, Gibco) supplemented with 10% FCS (Gibco) under antibiotic-antimycotic protection (AAS, Sigma 1:100). After triturating, the debris was removed by filtration through Millipore cell strainers (45 μm in diameter). Viable cells were plated at a density of 5×10⁴ cells/cm²on poly-L-lysine 24-well tissue culture plates in 500 μl DMEM supplemented with 10% FCS and AAS (1:100). The cells were maintained in a humidified atmosphere with 5% CO₂ at 37°C and allowed to grow for 7 days before CB-derived cells were added. Undifferentiated cells from nestin-positive clones were collected by trypsinisation and prelabelled with green `cell tracker' (5-chloromethyl-fluorescein-diacetate, Molecular Probes Inc), according to the manufacturer's recommendation. CB-derived cells were seeded on the monolayer of rat brain cells at a density of 5×10⁴ cells/cm². Cells were allowed to grow in such co-culture conditions for between 4 and 8 days and were then fixed for immunocytochemistry.

The cultures were harvested in PBS, counted and lysed in the Laemmli(Laemmli, 1970) gel loading buffer in the proportion of 3.4×10⁵ cells per 100 μl. The equal-volume samples were separated by SDS-PAGE on a 10% polyacrylamide gel and transferred onto Hybon-C-Extra. Immunodetection was performed using the monoclonal anti-β-tubulin III (Sigma), polyclonal anti-GFAP (DAKO) and polyclonal anti-PLP/DM-20 (gift from J.-M. Matthieu). The immunoblots were incubated with horseradish-peroxidase-conjugated secondary antibodies,anti-rabbit for GFAP and PLP/DM-20 antigens and anti-mouse for β-tubulin III detections, then developed by ECL (Amersham).

Cells were fixed with 4% paraformaldehyde diluted in PBS for 20 minutes,then washed with PBS and blocked in PBS containing 50% sheep serum and 10% FCS(60 minutes). First antibodies were applied overnight at 4°C. Anti-human nestin, a rabbit polyclonal antibody (gift of U. Lendahl, Karolinska Institute, Stockholm) was applied at a concentration 1:1000, according to Grigelioniene et al. (Grigelioniene et al., 1996). The three following primary antibodies, mouse monoclonal TUJ1 (Easter et al.,1993) directed against the β-tubulin isoform III (gift of A. Frankfurter, University of Virginia, Charlottesville, VA), mouse monoclonal anti-MAP-2 (microtubule associated protein 2, Sigma) and rabbit polyclonal anti-GFAP (glial fibrillary acidic protein, purchased from Dakopatts), were diluted 1:2000, 1:100 and 1:200, respectively, in PBS/gelatine containing 0.2%Triton X-100. The mouse monoclonal anti-GalC (galactosylceramide) antibody(Ranchst et al., 1982), a culture supernatant obtained from R-mAb hybridoma cells (gift of B. Zalc,INSERM U-495, Paris) was used at a dilution of 1:50 in DMEM with 10% FCS. Secondary antibodies, anti-mouse IgG FITC for MAP2 (Sigma), anti-mouse IgG2a-TxR for TUJ1, anti-mouse IgG3-TxR for GalC or anti-rabbit IgG-TxR for GFAP (all from Pharmingen), were diluted 1:100 in the same solution as the first antibody and applied for 1 hour at room temperature. As a control for immunocytochemistry (in order to exclude non-specific background staining),first antibodies were omitted during the procedure. To visualize the nuclei,the cultures were then incubated with 5 μM Hoechst 33258 (Sigma) (20 minutes at room temperature) before being mounted in Fluoromount-G (Southern Biotechnology Associate Inc., USA) either directly on the bottom of 24-well plates or on glass slides of poly-L-lysine-coated cover slips.

The live growing cells or prefixed immunocytochemically labelled cultures were observed either in the phase contrast or in the UV light under fluorescence microscopes using Axiovert 25 or Axioscope 2 (Carl Zeiss),respectively. Images were captured by the Videotronic CCD-4230 camera coupled with the microscope and processed using the computer-based programmable image analyser KS300 (Carl Zeiss).

The formation of clones by mitogen-expanded cells, which were selected from three independent cord blood preparations, was followed for at least four weeks for three or more randomly chosen clones. Differentiation towards a particular cell phenotype was quantified as a percentage of the total number of CB-derived cells growing in defined conditions. Cells from three culture plates (at least 600 cells each time) were counted in parallel for every cord blood preparation using the computer-assisted image analysis system described above.

Total RNA was isolated from cells using TRIzol Reagent (Life Technologies)and quantified spectrophotometrically. Then 5 μg samples were reverse transcribed using Superscript II and oligo (dT)_12-18 primers(Gibco). Each sample was amplified in duplicate, with and without reverse transcriptase, to control the amplification of genomic DNA.

An equal volume of each sample was amplified by PCR using the following primers: for the human nestin gene 5′-GAGGACCAGGACTCTCTATC-3′ and 5′-AGCGAGGAGGATGAGCTCGG-3′ and for the GAPDH gene 5′-CATGTGGGCCATGAGGTCCACCAC-3′ and 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′. Following 30 cycles of amplification (1 minute at 94°C, 1 minute at 58°C and 1 minute at 72°C using the MJ Research Thermal Cycler PTC-100), the PCR products were resolved on a 1% agarose gel. The appearance of 998 bp nestin bands was photographed under UV light.

CB-derived cells, which were negatively selected for hematopoietic (CD34)and endothelial (CD45) cell surface markers during 6 weeks of culture in the conditions given in Material and Methods(Fig. 1A,B), when treated with mitogens such as epidermal growth factor (EGF) display relatively high potency to expand (Fig. 2B). Then,after planting at a low-cell dispersion (approximately 10 cells per cm²), they grow clones. The clone-forming cells express nestin(Fig. 2E,F), a protein that was previously shown to be a reliable marker for central nervous system (CNS) stem and progenitor cells (Lendahl et al.,1990). Cell clones multiply at the rate of about 10 times the cell number per week (Fig. 2C,D),and after reseeding in the presence of EGF, they can grow further to form a monolayer of adherent, undifferentiated cells or, depending on the planting density, can re-establish new clones as observed up to six passages. Moreover,these new clones were totally unable to produce any hematopoietic colonies in standardised in vitro tests provided by Stem Cell Technologies (see Materials and Methods).

Differentiation of nestin-positive cells was achieved either by direct treatment of growing clones with differentiation-promoting media(Fig. 3A,B) or by plating clone-forming cells onto poly-L-lysine-coated coverslips in the presence of neurobasal/10%FCS medium. Cell differentiation was supported by addition of retinoic acid (RA) alone (Fig. 3A,B,C) or in combination with brain-derived neurotrophic factor(BDNF) (Fig. 4) as recommended previously by Sanchez-Ramos et al.(Sanchez-Ramos et al., 2000). Under these conditions the CB-derived cells start to differentiate along the three major CNS lines, which can be identified by their immunochemical properties. Cell-type-specific antigens were recognised by a TUJ1 monoclonal antibody directed against a neuron-specific class of III β-tubulin(Fig. 3A,B,Fig. 4A,B), by GFAP polyclonal antibody against an astrocyte-specific fibrillary acidic protein(Fig. 3A,Fig. 4C,D) and by a GalC monoclonal antibody against the oligodendrocyte-specific galactosylceramide(Fig. 3B,Fig. 4E,F) (for details see the Materials and Methods). Moreover, as is shown inFig. 3A,B, cells belonging to the same clone can express neuronal/astrocytic or neuronal/oligodendrocytic markers, confirming directly their dual differentiation potential. The appearance of the neural marker proteins upon CB cell differentiation was additionally proved by western blotting(Fig. 3C). A low level expression of the neuronal marker β-tubulin III can be found even in the initial, non-differentiated clonogenic cultures, whereas two other, astrocytic(GFAP) and oligodendrocytic (PLP/DM-20) markers, are detected only after growing the cells in differentiation-promoting conditions. For western blots,we have used, instead of a classic oligodendrocyte immunomarker, GalC, a proteolipid protein (PLP) and its splicing variant DM-20 expression. An early appearance of DM-20, which is known to overtake expression of PLP as well as GalC in the oligodendrocyte lineage, is clearly visible on the blot(arrow).

The degree of differentiation of CB-derived cells depends on supplementation of the medium (Fig. 6). Spontaneous differentiation after plating of the clone-growing cells on poly-L-lysine substratum in 10%-FCS-supplemented neurobasal medium was minimal for neurons and astrocytes (less than 5% of the whole cell population). Addition of RA into the medium promotes differentiation of neurons and, to a lesser extent, astrocytes. Supplementation of the medium with BDNF does not increase the number of neurons in comparison with RA alone,whereas it significantly promotes the development of astrocytes and suppresses that of oligodendrocytes. This may indicate that at this stage of differentiation of CB-derived cells (4 days after poly-L-lysine plating),neurons are not able to produce BDNF at a concentration that is optimal for its physiological paracrine effect on the neighbouring cells.

A similar or even higher differentiating effect was achieved after plating CB-derived cells on cover slips with already growing rat primary cortical culture in the presence of 10% FCS in DMEM medium. The phenotype-specific markers for neurons, astrocytes and oligodendrocytes (red inFig. 5B,F,J) co-stained numerous CB-derived cells that were pre-labelled with 5-chloromethyl-fluorescein-diacetate (green inFig. 5A,E,I). The close vicinity of rat-brain-differentiated cells appears to promote CB-derived cell differentiation. It seems that under these conditions the CB-derived neural precursors get an optimal paracrine neurotrophic support that promotes the appearance of all three types of neural progeny. After 4 days in co-culture,almost 40% of cells that were previously marked by green `cell tracker'differentiate into neurons, and for the other cells types 30% differentiate into astrocytes and 11% into oligodendrocytes(Fig. 6).

Fig. 7 presents the characteristic future of cells belonging to all three types of neural lineage. Some cells display typical neuron-like morphology, with long neurite projections. These cells express, in addition to TUJ1-labelled β-tubulin III, MAP-2 protein, which is characteristic of later steps of neuronal development. Overlaying images of cells double-labelled for these two neuronal proteins are shown in Fig. 7A. In Fig. 7B, distinct populations of anti-GFAP-reactive astrocytes (red) and anti-MAP2-stained neurons (green-labelled cytoskeletal structures) are shown to grow in proximity. Occasionally cells co-expressing both markers (GFAP and MAP-2) were observed; however this was seen in less than 2% of all the GFAP-labelled cells. Cells showing a typical morphology of matured, myelin-forming oligodendrocytes, with irregular, branched projections that stain with an anti-GalC antibody (owing to overlaying with the green `cell tracker' they are yellow in Fig. 7C) were often found in differentiating CB-derived cell cultures (Figs4 and5).

The CB-derived cells show a relatively high commitment to neuronal and astrocytic fate; a level similar to that previously observed with foetus-derived neural stem cells(Carpenter et al., 1999;Svendsen et al., 1999). Occasionally, in differentiating cultures a low level (about 2%) of cells co-expressing GFAP and MAP2 (being astrocytic and neuronal markers,respectively) was observed. This observation seems to match a recent finding that newborn neurons appearing in subventricular zone(Barres, 1999), as well as those differentiating from foetal stem cells in vitro(Rosser et al., 1997),co-express GFAP and probably originate from a certain type of common,neuro-astroglia progenitor.

Interestingly, neural cells obtained from CB show a relatively high spontaneous differentiation into oligodendrocytes, which amounts to about 11%of the cells in co-culturing conditions, a higher percentage than previously reported for CNS stem cells (Palm et al.,2000; Zhang et al.,2000).

Attempts are under way to test the responses of CB-derived cells to defined trophic or genetic signals, which are known to be effective in promoting differentiation of oligodendrocytes and neurons in vitro(Cameron et al., 1998;Josephson et al., 1998;Bużańska et al.,2001b) and to recruit them to the damaged brain in vivo(Fricker et al., 1999;Bjorklund and Lindvall, 2000;Rosser et al., 2000). Our data utilising a co-culture system as an alternative to in vivo injection studies indicate that brain tissue itself can provide optimal trophic support for neural progenitor cell differentiation.

The other challenge is to elucidate further the origin of the CB-derived neural precursors described here. We have already shown that this selected cell population, which is able to differentiate towards neural phenotypes, is practically devoid of cells expressing CD34 and CD45 antigens(Fig. 1B), which are characteristic of angiogenic or blood-forming stem cells(Kim et al., 1999). In this respect, their antigenic properties are similar to those described in the foetal human CNS stem cell subpopulation(Uchida et al., 2000). In contrast, the neural precursors examined for this study originate from a plastic adherent mononuclear fraction, which may suggest a mesenchymal origin. In spite of this, at the final stage of in vitro propagation and selection,which directly precedes nestin-expressing clone formation, these cells are totally unable to produce any hematopoietic colonies in vitro. This result corresponds with the antigenic properties estimated by FACS analysis in this paper (Fig. 1B). A similar fraction of plastic-adherent mouse bone marrow stromal cells was reported to transdifferentiate into a neural lineage by Kopen et al.(Kopen et al., 1999). As we have already shown, the CB-derived precursor cells can produce nestin-expressing clones that are able to differentiate toward neuronal/astrocytic or neuronal/oligodendrocytic phenotypes(Fig. 3A,B), thus displaying a bipotentiality. The question of whether these clones can differentiate simultaneously into all three types of neural progeny, a rigorous demand of a neural stem cells, must be answered in further experiments. It is also conceivable that CB-derived neural cells may originate from even more`primitive' pluripotent stem cells residing in cord blood and resembling those discovered recently in mouse bone marrow(Krause et al., 2001). These ancestor cells can differentiate in vivo toward a variety of cell types,including epithelial cells of the lung, gastrointestinal tract, liver, brain and skin. This striking potential for transdifferentiation of adult stem cells from various tissues into a neural fate as well as into cells of others organs(Kopen et al., 1999;Peterson et al., 1999) is a matter of increasing interest and discussion(Morrison, 2001). Thus, it will be scientifically and practically important to understand by which mechanisms the cells from cord blood give rise to developmentally unrelated CNS tissue and to further purify and characterise these cells.

In conclusion, this study has provided evidence, to our knowledge for the first time^*, that each of the three cell types of human brain — neurons, astrocytes and oligodendrocytes — can be propagated in vitro from CB cells. These results raise the possibility that cord blood may provide an efficient source of cells differentiating into the neural lineage, with a potential to be employed in the therapy of human CNS diseases.

We thank Anne Rosser for helpful suggestions, U. Lendahl and B. Zalc for their generous gifts of antiserea and Jan Albrecht for careful reading,discussion and editorial assistance. The work was supported by the grant No. 6P05 A04920 from the State Committee for Scientific Research and partially by the grant No. PBZ 29/12 from the Polish State Committee for Scientific Research and Ministry of Health.