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a Center of Excellence for Aging and Brain Repair and
b Departments of Neurosurgery,
c Neurology,
d Anatomy,
e Psychiatry,
f Pharmacology and Therapeutics, and
g Pathology, University of South Florida, College of Medicine, Tampa, Florida, USA;
h James A. Haley VA Hospital, Tampa, Florida, USA
Key Words. Human • Umbilical cord blood • Stem/progenitor cell • Multipotential differentiation • Hematopoietic and neural antigens • Neuropoiesis
Correspondence: Ning Chen, M.D., Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, College of Medicine, 12901 Bruce B. Downs Blvd. Tampa, Florida 33612, USA. Telephone: 813-974-7515; Fax: 813-974-3078; e-mail: nchen1{at}hsc.usf.edu
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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INTRODUCTION |
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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 [14–16] and delay both onset of symptoms and death of animals with amyotrophic lateral sclerosis [17, 18]. Furthermore, these cells were shown to be useful in models of traumatic brain injury [19, 20] and spinal cord injuty [21, 22]. In the Sanfilippo animal model of mucopolysacharidosis IIIB, histopathological improvements were found after HUCB administration [23]. In the clinic, treatment of a young Krabbe’s leuko-dystrophy patient with HUCB infusions resulted in attenuated progression of the disease [24]. These studies rely more on the trophic effect of the mononuclear HUCB cells than on actual cellular replacement. Only a few transplantation studies have addressed the possibility that the mononuclear fraction may contain a small number of nondifferentiated cells that may, under specific circumstances, such as stroke or placement into a favorable neurogenic environment [12, 15], give rise to neural-like cells.
Knowing that overlapping genetic programs for hematopoiesis and neuropoiesis exist [25], in this study we explored the possibility that stem/progenitor cells in the mononuclear HUCB fraction can produce progeny that express neural antigens when grown as adherent or nonadherent cultures. We used culture conditions commonly used for neural tissue and examined through available immunocytochemical stainings and Western blot analysis whether, and to what extent, the antigens typical for neural lineages would overlap with hematopoietic surface antigens characteristic for hematopoietic stem/progenitor cells or their fully differentiated progeny. In addition, we investigated whether the hematopoietic cells, expressing neural antigens without previous exposure to neuralizing epigenetic factors, would attain the morphology of neural-derived cells. These observations will help us clarify the real contribution of the mononuclear HUCB fraction to the overall pool of hematopoietic cells that seem to be able to transdifferentiate toward neural lineages or determine whether this transdifferetiation is just a temporary coexpression of hematopoietic and neural markers triggered by unusual or artificial environmental cues.
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MATERIALS AND METHODS |
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), and the cells were categorized based on their size and morphology [26].
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Adherent Culture Cryopreserved samples of HUCB mononuclear fraction (supplied by Cambrex Corporation, East Rutherford, NJ, http://www.cambrex.com, or Saneron CCEL Therapeutics, Inc., Tampa, FL, http://www.saneron-ccel.com/pages/893814/) were thawed rapidly in a 37°C water bath, suspended in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) with 10% fetal bovine serum (FBS) (Invitrogen) and 0.1% gentamicin (Sigma-Aldrich) in a 15-ml conical tube (Falcon, BD Biosciences, San Diego, http://www.bdbiosciences.com), centrifuged (400g/7 minutes), and resuspended in 1 ml of the above-mentioned fresh medium. Cell viability (62%–90%) was assessed by a rapid staining procedure with 0.4% try-pan blue (Invitrogen). Cells were plated in the described medium on poly-3-lysine-coated (PLL) (10 µg/ml) eight-well chamber slides (Lab-Tek, Campbell, CA, http://www.labtek.net/) at a seeding density of 100,000 cells per cm2. All cultures were incubated at 37°C in 5% CO2 for 1–20 days in vitro (DIV), with fresh medium added every 5 days. Four donor samples were used in this experiment. Experiments were performed in duplicate for each time point.
Floating HUCB Fraction Several cultures (20–34 DIV) with whole mononuclear HUCB cells were used to generate the floating population. The supernatants were collected, cells were spun down (400g/7 minutes), and the viability of the aliquot was determined (95%) using 0.4% trypan blue. The cells were replated into fresh DMEM (containing 10% FBS and 0.1% gentamicin) on PLL-coated (10 µg/ml) 24-well plates (Nunc, Rochester, NY, http://www.nuncbrand.com) at a seeding density of 100,000 cells per cm2. Fresh medium was added on day 5. The cultures were fixed and analyzed on day 10.
Dissection and Culture of Embryonic Tissue
Sprague-Dawley timed-pregnant rats (Harlan, Indianapolis, http://www.harlan.com) were anesthetized with Equithesin (3 ml/kg, i.p.), and the embryonic (E14-18) brains (15 embryos per dam) were removed from their skulls. The cortical and olfactory bulb [27, 28] tissues were dissected and mechanically dissociated in Hanks’ balanced salt solution/HEPES solution with 0.05% DNase. The cells were resuspended in DMEM, 10% FBS, and 0.1% gentamicin and plated on PLL-coated 24-well plates at a seeding density of 75,000 cells per cm2. Preplating viability ranged from 90%–95%. A day after plating, the medium was changed to Neurobasal (Gibco, Grand Island, NY, http://www.invitrogen.com) with 0.01% of B27 supplement (Gibco) and gentamicin. The cells were maintained at 37°C in 5% CO2 with 95% humidity for up to 10 DIV.
Viability of Cultured HUCB Cells
Before fixation, fluorescein diacetate/propidium iodide (FDA/PI) (Sigma-Aldrich) staining was used to assess the viability of the adherent mononuclear fraction at 1, 5, 10, 15, and 20 DIV. The numbers of living (green) and dead (red) cells were counted in 16 randomly selected fields per chamber slide per time point under an inverted fluorescent microscope. Cell viability was calculated as the number of living cells divided by the total number of cells multiplied by 100.
Isolation of CD133+ Cells from HUCB Mononuclear Fraction
Cryopreserved HUCB mononuclear fractions purchased from Cambrex Corporation were thawed rapidly in a 37°C water bath and then washed with phosphate-buffered saline (PBS) containing 2 mM EDTA (Sigma-Aldrich). The cell pellet was resuspended in a final volume of 300 µl of buffer (PBS, pH 7.2, supplemented with 0.5% bovine serum albumin and 2 mM EDTA) per 107 total cells, and then the CD133+ cells were separated out using a CD133 cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) according to manufacturer’s instructions. Briefly, after adding 100 µl FcR blocking reagent, cells were incubated with CD133 MicroBeads for 30 minutes at 4°C to 8°C. CD133+ cells then were isolated from the HUCB mononuclear fraction by a Mini MACs (a magnetic cell separator; Miltenyi Biotec). After separation, the total number of positive cells and the viability were determined using a hemacytometer. A total of 3.1 x 104 CD133+ cells was isolated from the HUCB mononuclear fraction, and viability was 98%. Immunocytochemical staining confirmed all isolated cells were positive for CD133.
Immunocytochemistry
Immunocytochemistry was used for identification of hematopoietic, embryonic, and neural-specific antigens in HUCB cells and primary embryonic cell cultures. The HUCB cultures (adherent and floating) were fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4) at 1, 5, 10, 15, and 20 DIV, whereas primary cell cultures were fixed at day 10. After rinsing three times in PBS, the cells were blocked with 10% goat serum and 0.1% Triton X-100 (omitted for immunolabeling of surface markers) in PBS for 1 hour at room temperature (RT). They were then incubated with the primary antibody (Table 2) at 4°C for 24 hours, washed in PBS three times, and incubated with a species-appropriate secondary antibody (Alexa Fluor 594, 488; Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com; 1:800 and 1:500, respectively) for 1 hour at RT, washed again with cold PBS, and coverslipped with 95% glycerol. Cell nuclei were visualized with 4,6-diamidino-2-phenylindole hydrochloride (100 ng/ml; Sigma-Aldrich). All culture slides were examined under BX-60 or IX-71 microscopes (Olympus, Tokyo, http://www.olympus-global.com).
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). To estimate the number of cells positive for hematopoietic, embryonic, and neural markers, the number of immunopositive cells and the total number of cells were counted using fluorescent microscopy by a single individual. The percentage was calculated as the number of cells positive for the specific antigen divided by the total number of cells and then multiplied by 100. For each antigenic marker, counts were taken from five randomly selected fields in the well at x 20 magnification and expressed as mean ± SEM. The total number of counted cells for each antigen ranged from 1,500 to 2,500. Analysis of variance was used to determine the differences in the number of cells expressing the specific antigen at each of the different culturing periods (1, 5, 10, 15, and 20 DIV). The criterion of significance was set at p < .05.
The staining intensity of each antigen was also assessed quantitatively using a 0–4 ordinal scale, in which 0 = absent, 1 = minimal, 2 = moderate, 3 = high, and 4 = maximal. The average degree of immunostaining was then calculated for each antigen in five fields.
To compare the two subpopulations (adhered and floating) of HUCB cells, statistical analysis of the data was performed using the program GB-STAT version 7.0 (DynamicMicrosystems, Inc., Silver Spring, MD, http://www.gbstat.com) by Student’s t-test with a significance level of p < .05 (two-tailed).
Western Blot
HUCB cells were cultured for either 5 or 10 DIV. Positive controls were prepared from male Sprague-Dawley rat brains (5 months old) euthanatized by decapitation. Longer-term cultures of the floating replated HUCB population (190 days), which highly expressed nestin and TuJ1, were also used as a positive control. The cell pellets or harvested brain tissue samples were suspended in the homogenization buffer (pH 8.0). The samples were homogenized by ultrasound and centrifuged (800g/20 minutes) to remove the nuclear fraction, and the resulting supernatants were collected. Protein concentration (for each sample) was assayed by bicinchoninic acid (Pierce, Rockford, IL, http://www.piercenet.com). The defined amount of protein (50 µg/40 µl) was separated by SDS-PAGE on a 7%–10% polyacrylamide gel, transferred on nitrocellulose membranes, and incubated for 1 hour at RT with 5% nonfat powdered milk in Tris-buffered saline containing 0.1% Tween 20 (TTBS) (pH 8.3). Immunodetection was performed using a monoclonal antibody against Nestin (1:100; BD Biosciences), TuJ1 (1:1,000; Covance, Princeton, NJ, http://www.covance.com), A2B5 (1:200; Chemicon, Temecula, CA, http://www.chemicon.com), or GFAP (1:500; Harlan) overnight at 4°C. After washing in TTBS, a secondary antibody conjugated to alkaline phosphatase (1:3,000; Bio-Rad, Hercules, CA, http://www.bio-rad.com) was applied for 1 hour at RT, and then the sample was washed again with TTBS. The reaction product was detected using a chemiluminescent protein detection system (Bio-Rad).
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RESULTS |
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). Two samples from four donors each were used. The stained cells were categorized into lymphoid (54%) and myeloid (7%) lineages based on the appearance of their nuclei and cytoplasm. The cells from the lymphoid lineage consisted of lymphoblasts (Fig. 1A, part A’ [H]) and small lymphocytes (Fig. 1A, part A’ [I]). Cells of the myeloid lineage had a characteristic granular cytoplasm (granulocytes) and consisted of monocytes (Fig. 1A, part A’ [F, G]) and myeloid (Fig. 1A, part A’ [B–E]) cells. Thirty-nine percent of the cells were not as easily defined. The morphology of these cells was round and similar to lymphocytes. Additionally, 1%–2% of cells resembled immature thrombocytes and erythrocytes. The number of mature erythrocytes was negligible (0.78%, Table 1).
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), viability was maintained at 50%–60%. The morphology of plated cells changed rapidly over time (Fig. 1A [C]). These initially small, round cells attained a new morphological appearance within 24–48 hours after plating. Numerous cells within clusters started to send out tiny processes. Later on (5–20 DIV), clearly distinct cell types appeared (Fig. 1B). The large egg-shaped cells without processes were common. They tightly adhered to the culture plate and frequently had numerous small cells on their surface or in close vicinity to the cell. The most frequently observed cell type was the small, round cell, either with one or two processes of variable length or with a ciliated surface.
HUCB Cells Reveal Immunoreactivity for Hematopoietic and Neural Antigens
We cultured HUCB mononuclear cells with serum-containing medium because these cells grow better in serum. The same lot of serum was used throughout the study, which allowed us to compare the morphology and cell type–specific antigens across several platings and time points (1, 5, 10, 15, and 20 DIV). Under these conditions, we found that there were two subpopulations of cells in the HUCB mononuclear fraction, an adherent and a floating population. Cellular composition, morphology, viability, and developmental potential of these two subpopulations are different.
Adherent Monolayer Cultures
Expression of Hematopoietic Antigens
We found that most adherent HUCB cells (90.85% ± 3.33%) expressed common leukocyte antigen (CD45) at all examined time points (Fig. 2A [A]). The morphology of these CD45+ cells was generally round without differentiation. Only a few cells expressed CD133 (2.72% ± 1.88%; Fig. 2A [C]) and CD117 (6.67% ± 4.23%) on day 1 (Fig. 3A). Both of these antigens were used for identifying stem/progenitor cells. These cells were small and round and were scattered throughout the culture. In addition, we noted that only a small number of CD15+ cells were present in this subfraction over all time points, although expression peaked at 10 DIV (18.13% ± 5.95%; Fig. 2A [B]). CD15 is normally expressed on 90% of human circulating granular cells and 30%–60% of circulating monocytes. It is also known as stage-specific embryonic antigen (SSEA-1) or Lewis X because it recognizes the carbohydrate epitope 3-fucosyl-N-acetyl-lactosamine on embryonic stem cells.
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Expression of Early and Mature Neural Antigens
To explore the potential of the adherent HUCB cells to produce neuron-like cells, we investigated the expression of neural proteins in these cells. Cells were immunofluorescently labeled with specific antigens, and the percentage of cells expressing each antigen was determined at all examined time points during the study (Figs. 2, 3). Many antigens typically found in the brain were expressed in the adherent fraction of HUCB cells. The highest frequency of expression was observed at 10–15 DIV. Nestin expression increased from 1.9% ± 1.3% after 1 DIV to 33.44% ± 11.51% after 15 DIV (Fig. 2A [D], Fig. 3B). Similarly, A2B5 (Fig. 2A [E]) expression peaked (28% ± 4%) at 15 DIV and decreased to 1.59% ± 0.46% by 20 DIV. In addition, regardless of their morphology and the length of the culturing period, almost all cells (average, 95.55% ± 2.3%) expressed vimentin, which is not only a marker of early neural cells but also a marker of mesenchymal origin. Besides these three antigens which are indicative of immature cells, we also detected mature glial antigens. GFAP was highly expressed in round cells at all times except 20 DIV, when expression was negligible (Fig. 3C) and strictly confined to small cells with two processes (Fig. 2A [J]). At the same time, S100 was highly expressed by many medium-sized cells. Further, the oligodendrocyte marker galactocerebroside was detected on the surface of round and irregularly shaped cells (Fig. 2A [K]) throughout the entire culture period.
HUCB cells within the adherent fraction in our study conditions were less likely to express either immature or mature neuronal markers. Immature neuronal antigen TuJ1 was present in a small population of round-shaped cells or cells with single processes at 1, 5, and 10 DIV (Fig. 2A [G]), although expression was highest at 1 DIV (7.9% ± 5.9%). We did not find TuJ1+ cells at 15 or 20 DIV. The mature neuronal antigen, MAP2, was almost absent in adherent HUCB cells (Fig. 3C). Those rare MAP2+ cells, however, had a neuron-like morphology with small somas and long, thin processes (Fig. 2A [I]). Interestingly, NF68KD expression was found in 34.86% ± 10.14% of the cells at all studied intervals (Fig. 3C). NF68KD predominantly stains neurons of the central and peripheral nervous system. This antigen was mostly expressed in cells with multiple processes (Fig. 2A [H]).
To elucidate whether there is coexpression of hematopoietic and neural antigens within the same cells, we first labeled adherent cultures with the antibody recognizing CD45 and afterward labeled cells with TuJ1, MAP2, or GFAP (Fig. 2B). Some HUCB cells coexpressed CD45 antigen and TuJ1 (Fig. 2B [A]; CD45+/TuJ1+) or GFAP (Fig. 2B [C]; CD45+/GFAP+). Interestingly, CD45 was not coexpressed with MAP2 (Fig. 2B [B]), and the morphologies of these CD45–/MAP2+ cells were different from the CD45-positive cells. MAP2 immunoreactive cells (CD45–/MAP2+) were well-differentiated, extending long processes. We also observed positive cells with coexpression of vimentin and GFAP antigens (vimentin+/GFAP+).
Presence of Neurotrophin and Chemokine Receptors
In this study, we also investigated the expression of neurotrophic receptors in the adherent HUCB subfraction, including the high-affinity receptors (trkA, trkB, and trkC) and low-affinity receptor p75NTR. The presence of neurotrophin receptors suggests that these cells could be induced into becoming neural cells. We noted that most of the HUCB cells in the adherent cultures expressed trkB and trkC. The trkA and p75NTR receptors, however, were rare in the adherent fraction (Fig. 3D). From 5 DIV, trkB was detected and reached a peak at 15 DIV (90.44% ± 2.86%; Fig. 2A[M]; Fig. 3D), whereas trkC was expressed on round cells at all time points (Fig. 2A[N]; Fig. 3D). In this experiment, we also examined the expression of CXCR4, the stromal derived factor-1 (SDF-1) receptor; SDF-1 is a chemokine important in migration of hematopoietic cells as well as in the development of the central nervous system (CNS). Half of the cultured HUCB cells expressed this antigen, although the morphology of these CXCR4+ cells was varied (Fig. 2A [O]).
Replated Floating Mononuclear HUCB Fraction
Morphology and Phenotype
We collected the floating population from 20- to 34-DIV HUCB cultures. Viability of these cells was high (90%–95%). After replating, these cells attached and practically all cells began to differentiate within 24–48 hours (Fig. 1D). The major difference in morphology between the replated floating fraction and the adherent fraction was that there were no large, egg-shaped cells in the replated floating fraction. In addition, there were numerous cells with long, thin processes in this fraction.
When we compared the incidence of hematopoietic antigens presented in both the adherent and floating-replate fractions, we found substantially fewer CD45-labeled cells and a large number of immature cells expressing CD133 and CD117 surface markers (Table 3) in the replated floating fraction; further, there was a significant increase in the number of cells expressing A2B5 (p < .001) and both trkB and trkC (p < .05) and a significant decrease in all mature glial markers (p < .05 to .001). This finding suggests that there may be more stem/progenitor cells present in the replated floating fraction that could be induced into a neuronal lineage. Even so, when we separated the CD133+ cell population from the whole mononuclear fraction, these cells failed to proliferate under the same culture conditions used with both the adherent and floating fractions. Although they maintained their cellular morphology throughout the study period, all of the cells died within 3 weeks of culturing.
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; Table 4).
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DISCUSSION |
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Basic Characteristics of the HUCB Cells In Vitro
The freshly thawed HUCB mononuclear fraction was comprised of 53% mature lymphoid cells. Only 1.95% and 3.1% of HUCB mononuclear cells were myeloid cells or monocytes, respectively, and most of these were more immature than those reported for bone marrow [29]. These immature cells in HUCB may actively proliferate and have multipotential differentiation capabilities when cultured in the absence of extrinsic promoting factors [30]. Mayani et al. showed that survival and proliferation of these cultured cells depended on high seeding density (1 x 107 to 1 x 108/cm2). This may be because HUCB cells cultured at higher seeding densities contain a larger number of stromal precursors, which are able to develop and form a feeder layer (our putative adherent subpopulation); this layer in turn generates sufficient intrinsic promoting factors such as stem cell factor and interleukin 6 [31] to support long-term survival of the nonadherent HUCB population (our floating-replate subpopulation).
The cultured HUCB mononuclear fraction was heterogeneous, with six common morphological cell types (Fig. 1C). The two most common in our observations were the large egg-shaped cells and the small round cells. The large egg-shaped cells that attached tightly to culture vessels and preferred serum-containing medium were part of the adherent subfraction. This feeder layer has been described by Ye et al. [31], who demonstrated that the central components of this layer were fibroblasts, macrophages, endothelial cells, and several extracellular matrix proteins. The small round cells sit on the top of large egg-shaped cells or were floating in the medium and hence were part of the floating-replate population; these cells were highly proliferative and multipotent. The morphology of these cells changed over time in culture.
Expression of Hematopoietic and Neural Antigens
Fully 90% of the adherent HUCB cells expressed the common leukocyte antigen CD45. In addition, there were few cells in this fraction that transiently expressed stem/precursor antigens (CD133 and CD117). More cells that expressed early stem cell antigens were found in the floating-replated population of HUCB cells and not the adherent cell subpopulation. In our culture, these CD133+ cells seem to be quiescent because we did not see any changes in morphology or proliferation in purified cultures of CD133+ cells maintained under the same conditions as we used for the floating or adherent cultures. This is consistent with the hypothesis that adult stem cells exist in a quiescent state in the blood until an injury or disease state triggers the stem cells to proliferate or differentiate through the production of specific factors [32].
Another stem cell antigen, CD15+, or SSEA-1, was found in the HUCB cell cultures. This antigen is expressed on embryonic stem cells, primitive hematopoietic stem cells, and also within the developing human brain [33, 34]. The expression of CD15 antigen in both hematopoietic and neural lineages suggests that there may be a set of overlapping genes controlling genesis in both lineages.
The expression of neurotrophin receptors in HUCB cells certainly suggests that the HUCB cells could be driven to a neural fate. By binding to their appropriate receptors, neurotrophins play important roles in differentiation and survival of neural cells during CNS development. The activity of trk receptors themselves also affects cell survival by adenosine [35]. Although the large number of trkB+ and trkC+ cells observed in cultured HUCB cells indicated that cells are responsive to brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4/5 and may be instrumental in transdifferentiation of the HUCB cells into neural cells, there is evidence that the neurotrophins play a functional role in the peripheral immune response. Neurotrophins are expressed by platelets [36] and B cells [37] and mediate activation and survival of eosinophils in allergic bronchial asthma [38]. They also enhance cytokine mRNA expression [39] in blood cells.
The CXCR4 chemokine receptor for SDF-1 is associated with normal development of the nervous system and is widely distributed throughout the body. SDF-1 induces leukocyte and hematopoietic progenitor migration through interactions with CXCR4; SDF-1 and a gradient of chemokine ligands also direct migration of the primordial germ cells [40, 41]. The deletion of CXCR4/SDF-1 genes in mice resulted in fetal mortality with abnormal cerebellar development [42]. Although this chemokine is expressed in response to injury or disease, which may be useful for the development of the HUCB cells as a therapeutic agent, SDF-1’s role in the neural development is not yet completely understood.
The presence of stem cell antigens within the HUCB population that we have examined suggests that there is a stem/progenitor population that may give rise to both hematopoietic cells and cells normally derived from other germ cell layers (e.g., neural cells) under the appropriate conditions. Certainly, when we examined the expression of neural proteins in HUCB cultures, many of the cells were positive. All of the neural antigens chosen (nestin, vimentin, A2B5, TuJ1, MAP2, GFAP, S100, NF68KD [NFL], and neurotrophin receptors) were observed within the HUCB subpopulations. We observed more glial antigens expressed in the adherent population and more neuronal antigens in the floating population. Our results are consistent with our earlier observations [13] and the observations of other laboratories [10, 11] and could support an argument for transdifferentiation. However, without a functional assay, alternative explanations are more likely. We observed coexpression of both hematopoietic and neural antigens in some cells. In vivo, the coexpression of two different lineage markers could be indicative of cell fusion with the host cells, but this could not apply to our culture dish, in which only HUCB cells were plated. An alternative explanation is that mononuclear cells do express some of these antigens normally. GFAP is found in some cells within the bone marrow compartment of the hematopoietic system [43]. A third possible explanation was elaborated by Lu et al. [44], who demonstrated that transdifferentiation of mesenchymal stem cells from bone marrow into neural cells is more likely a function of cellular toxicity with resulting changes to the cytoskeleton and not a change in the cellular differentiation pathways. Clearly the best demonstration that the cells have attained a neural phenotype will be through functional assays showing not only a neural morphology and protein expression but also synapse formation, neural electrophysiological activity, and neurotransmitter release. Even if we can demonstrate the true conversion of the HUCB cells into neural phenotypes, in vitro expansion of the cells may be problematic, as this may silence the cells’ capacity to differentiate and engraft [45]. Further studies are warranted to better understand how to best use these cells in the development of cell therapies to restore brain function.
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ACKNOWLEDGMENTS |
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DISCLOSURES
P.R.S. is cofounder and owner of Saneron CCEL Therapeutics, Inc. A.E.W., S.G.D., T.Z., and J.S.R. are consultants to Saneron CCEL Therapeutics, Inc. P.R.S., S.G.D., J.S.R., and A.E.W. are coinventors on HUCB-related patents.
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