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江边孤钓

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发表于 2009-3-5 10:47 |只看该作者 |倒序浏览 |打印

a Center of Excellence for Aging and Brain Repair and! l: _  Q0 Y8 ~  E! L! v0 r* C" f
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b Departments of Neurosurgery,
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c Neurology,: j' _, D5 h3 D0 _, u

d Anatomy,  T, m6 [  W# c2 w' z2 _# k( `

e Psychiatry,
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f Pharmacology and Therapeutics, and

g Pathology, University of South Florida, College of Medicine, Tampa, Florida, USA;
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h James A. Haley VA Hospital, Tampa, Florida, USA  L# D$ Z8 M1 g+ L& E0 @- C' }6 x
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Key Words. Human ? Umbilical cord blood ? Stem/progenitor cell ? Multipotential differentiation ? Hematopoietic and neural antigens ? Neuropoiesis$ v; |5 q4 V0 ~
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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@hsc.usf.edu
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ABSTRACT

Human umbilical cord blood (HUCB) cells have been used as a powerful tool for the treatment of several blood-related diseases such as Fanconi anemia , leukemia , thalassemia, sickle cell disease , and Wiscott-Aldrich syndrome . Recently it was observed that cells derived from this source have the ability to express markers and morphologies of other cell types within the same mesodermal germ layer such as bone , fat , smooth muscle , and skeletal muscle . More surprisingly, exposing HUCB cells to various experimental conditions showed that their progeny could also reveal properties typical of neuroectoderm-derived cells . This multilineage differentiation capacity and the expression of neural properties suggests that HUCB cells may have the ability to transdifferentiate or become nonhematopoietic cells of various tissue lineages, including neural cells. If this is the case, they may therefore be useful for numerous cell-based therapies requiring either the replacement of individual cell types or substitution/replenishment of missing substances.! t& Z' K( x2 c( a6 {& t, H: g
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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  and delay both onset of symptoms and death of animals with amyotrophic lateral sclerosis . Furthermore, these cells were shown to be useful in models of traumatic brain injury  and spinal cord injuty . In the Sanfilippo animal model of mucopolysacharidosis IIIB, histopathological improvements were found after HUCB administration . In the clinic, treatment of a young Krabbe’s leuko-dystrophy patient with HUCB infusions resulted in attenuated progression of the disease . 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 , give rise to neural-like cells.
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Knowing that overlapping genetic programs for hematopoiesis and neuropoiesis exist , 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.

MATERIALS AND METHODS/ C. h. g! r) ?0 j, ?. N" Y; l
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Cellular Composition of the Mononuclear HUCB Fraction Before Culturing
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Upon thaw of the HUCB mononuclear cells, Wright-Giemsa smears were prepared to determine the ratio of differentiated and undifferentiated leukocytes (Fig. 1A). 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’ ) and small lymphocytes (Fig. 1A, part A’ ). Cells of the myeloid lineage had a characteristic granular cytoplasm (granulocytes) and consisted of monocytes (Fig. 1A, part A’ ) and myeloid (Fig. 1A, part A’ ) 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).

Figure 1. The Wright-Giemsa staining and morphology of a mononuclear HUCB fraction before and after culturing. (Panel A): Part A, Giemsa stained smear of HUCB cells. Scale bar = 10 μm. Part A’ shows high magnification of individual cell types depicted on the previous image: (B) myeloblast, (C) promyelocyte, (D) band, (E) neutrophil, (F) promonocyte, (G) monocyte, (H) lymphoblast, (I) small lymphocyte, and (J) orthochromic normoblast. Scale bar = 2 μm. Part B shows viability of cultured mononuclear HUCB cells; living cultures were stained with fluorescein diacetate/propidium iodide to detect healthy (green) and dead (red) cells. Part C shows bright-field photomicrograph of Giemsa-stained cultured HUCB cells (6 DIV) demonstrating heterogeneous morphologies (arrows). Scale bar = 20 μm. (Panel B): Morphology of the adherent population of mononuclear HUCB cells cultured for 20 days. (A): One day after plating, cells (arrowheads) formed small clusters from which a few cells sent out tiny processes (arrows). During the next culture period (5–10 days), the clusters became loosely packed and many cells extended longer processes interconnecting cell groups. (B, C): Besides small round cells either with or without processes (arrows), numerous larger round cells (asterisks) were found. These cells were always tightly adhered to the bottom of the culture well. (D, E): In the next two time periods (15 and 20 DIV), small cells (arrowheads) with longer processes were interspersed either with individual or groups of round flat cells (asterisks). Scale bar = (A) 10 μm and (BE) 20 μm. (Panel C): Different cell types were observed in adherent cultures: (A) large egg-shaped cells; (B) bipolar and (D) multipolar cells; (E) small round cells with multiple hair-like spines; and small cells with (C) thicker or (F) thinner processes. Scale bar = 2 μm. (Panel D): The morphology of cultured HUCB cells prepared from floating fractions. Morphologically, the cells were similar to the adherent population, with a striking absence of big round egg-like cells. (A): Numerous clusters (arrow) of uniform round cells with occasional processes were observed. (B): Higher magnification of the boxed area from (A). Scale bar = (A) 40 μm and (B) 20 μm. Abbreviations: DIV, days in vitro; HUCB, human umbilical cord blood.. S$ W! {/ t- j) j4 G1 `
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Viability and Morphology of Cultured Mononuclear HUCB Cells$ B6 g. |: u/ v8 l  v

The viability of the HUCB cells at thaw ranged from 65%–85%. After 5 DIV, FDA/PI fluorescent staining revealed that viability remained high (85%), whereas at later time points (10, 15, and 20 DIV; Fig. 1A ), viability was maintained at 50%–60%. The morphology of plated cells changed rapidly over time (Fig. 1A ). 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.  a6 n  i* p! V; s

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.3 f( G6 {. J8 S
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Adherent Monolayer Cultures/ e/ |9 Z% Q1 C! Z

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 ). The morphology of these CD45  cells was generally round without differentiation. Only a few cells expressed CD133 (2.72% ± 1.88%; Fig. 2A ) 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 ). 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.( }5 Q6 }3 L# i% U
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Figure 2. Fluorescent images. (Panel A): Immunocytochemical characterization of (A–C) adherent mononuclear HUCB cells showing the expression of hematopoietic CD antigens. (A): Almost every cell revealed surface CD45 (green) antigen at 1, 5, 10, 15, and 20 days. (B): Only a few cells were positive for the antibody against CD15 (green, arrows) throughout the entire course of the study. (C): The presence of immature progenitors expressing CD133 (green, arrow) was recorded only in short-term (1 DIV) cultures. DAPI counterstaining was used for visualization of the entire cell population. Scale bar = (A, B) 20 μm and (C) 10 μm. (D–F): Several early neural markers were expressed in HUCB cells. Cells positive for (D) nestin, (E) A2B5, and (F) vimentin were observed throughout the entire culturing period. Arrows in (D–F) point to immunoreactive cells. (G–I): HUCB cells expressed early and mature neuronal antigens. Ten days after culturing, only a few cells expressed TuJ1 (G, arrows) and MAP2 (I). The arrow points to the MAP2-positive soma, whereas arrowheads delineate the long process. (H): At the same time, many cells were immunoreactive for NF68KD (arrows). Scale bar = (D–I) 20 μm. (J–L): Cultured HUCB cells express glial antigens. (J): Numerous cells were positive for GFAP. The arrows point to two cells with bipolar morphology. In sister cultures, the positivity for other glial markers such as S100 (K, arrows) and GalC (L) was detected. Scale bar = (J, K) 20 μm and (L) 10 μm. (M–O): Expression of neurotrophin and chemokine receptors. (M): Positivity for trkB was found on day 5 only, whereas trkC (N, arrows) was present throughout the whole culture period. (O): CXCR4 was present in numerous cells in all studied intervals (arrows). Scale bar = (M–O) 10 μm. (PanelB): Coexpression of hematopoietic and neural antigens in cultured HUCB cells. (A): Several CD45-positive (green) cells coexpressing TuJ1 (red). Hematopoietic CD markers are localized on the cell surface (arrows) of many HUCB cells, whereas weak cytoplasmic TuJ1 expression was found occasionally. Nuclear DAPI labeling (blue) confirms that not every HUCB cell expresses CD antigens. (B): For longer cultures, a cell with distinct processes positive for mature neuronal marker MAP2 (red) and negative for CD45. On the other hand, cells with round morphology were CD45 immunoreactive (green). (C): Coexpression of GFAP (red) and CD45 (green) was also found (arrows). (D): HUCB cells were immunopositive for vimentin (green), and some also expressed GFAP (red). The arrow indicates vimentin/GFAP-postive cells. Inset shows a double-labeled vimentin/GFAP-positive (orange) cell at a higher magnification. Scale bar = (A–C, inset in D) 10 μm and (D) 20 μm. (Panel C): Fluorescent images of replated floating HUCB cells. (A–C): Hematopoietic markers, including (A) CD45, (B) CD133, and (C) CD117. Arrowheads point to cells negative for the specific marker, whereas arrows indicate cells expressing one of the CD antigens. (D, E): Expression of (D) nestin and (E) A2B5 (arrows) was higher in the replated floating fraction than in the adhered HUCB cells. (F–I): Cells positive for (F) TuJ1, (G) GFAP, (H) S100, and (I) GalC. Morphologically, this population of cells rarely acquired the classical appearance of neural derivatives. (J, K): Expression of neurotrophin receptors (J) trkB and (K) trkC was also detected. Arrows in (F–K) point to positively labeled cells. Blue DAPI counterstaining was used for clear identification of all cultured cells. Scale bar = (A, D) 20 μm and (B, C, E–K) 40 μm. (Panel D): Expression of neural antigens in E14-E18 primary cultures. (A–C): Cells isolated from the embryonic striatum were immunopositive for (A) nestin and (B) vimentin, and cultures prepared from the embryonic olfactory bulb revealed positivity for (C) A2B5. Scale bar = (A) 10 μm and (B, C) 40 μm. TuJ1 (D) and MAP2 (E) were found in cortical cultures after 10 days. Cells expressing these two neuronal antigens revealed classical neuronal morphology. (F): Small neuronal bodies and long, branching processes differed from flat, larger cells expressing GFAP. Inset shows the detailed morphology of GFAP-positive cells taken from E18 striatal culture. Scale bar = (D) 40 μm, (E) 10 μm, and 20 μm. Scale bar in inset = 10 μm. (G, H): Cortex-derived cells also expressed two other glial antigens. Arrows in (G) point to S100-positive cells. Scale bar = 20 μm. (I, J): Cortical cells also reveal positivity for neurotrophin receptors. Scale bar = 10 μm. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; DIV, days in vitro; HUCB, human umbilical cord blood.- d% l5 _* L* E
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Figure 3. Antigen expression in adherent HUCB cells cultured in serum from 1–20 days in vitro. (A): Expression of early hematopoietic and stem cell antigens. (B): Expression of early neural/stem cell antigens. (C): Expression of antigens indicative of immature and mature neurons, astrocytes, and oligodendrocytes. (D): The adherent HUCB cells also expressed both neurotrophin receptors and the receptor for the chemokine stromal-derived factor 1, CXCR4. Abbreviation: HUCB, human umbilical cord blood.

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 , Fig. 3B). Similarly, A2B5 (Fig. 2A ) 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 ). 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 ) throughout the entire culture period./ j8 _7 \: Q" Y+ I) r4 [# J
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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 ), 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 ). 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 ).) J6 L1 I# T6 N' \+ V' h
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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 ; CD45 /TuJ1 ) or GFAP (Fig. 2B ; CD45 /GFAP ). Interestingly, CD45 was not coexpressed with MAP2 (Fig. 2B ), 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; Fig. 3D), whereas trkC was expressed on round cells at all time points (Fig. 2A; 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 ).
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Replated Floating Mononuclear HUCB Fraction5 r9 d! U0 I$ a' y5 c6 X

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
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Table 3. Incidence of hematopoietic and neural antigens in both populations of cultured HUCB cells
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We also used adult rat brain (5 months old) as a positive control for CNS antigen expression, degree of immunofluorescent labeling, and true neural morphologies (Fig. 2D; Table 4).& H' f2 }( o) }7 O& m6 Q  z
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Table 4. Expression of various antigens by both (adherent and floating-replated) HUCBs and primary cultures (E14-18 embryonic rat brain)

Western Blot
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For Western blots, the following cell preparations were examined: HUCB mononuclear cells on thaw (HUCB day 0), the adherent HUCB cells cultured for 5 or 10 DIV; primary cultures from 5-month-old rat brain (positive control); and floating-replate HUCB cells (cultured for 190 DIV and having extensive TuJ1 expression). Western blot analysis confirmed immunocytochemical detection of the neural markers nestin, TuJ1, A2B5, and GFAP. Adherent HUCB cells showed a weak intensity band for nestin at 5 and 10 DIV (220 KD), whereas the floating-replate HUCB cells were strongly positive for nestin; we did not find nestin in the 5-month-old rat brain preparation, most likely because nestin is commonly present only during early development of brain. TuJ1 (50 KD) was weakly expressed in HUCB cells at thaw and at 5 and 10 DIV, whereas the floating-replate subpopulation and the rat brain had strong TuJ1 immunolabeling. Weak bands were seen for A2B5 (42 KD) and GFAP (56 KD) in all samples. The ?-actin loading control demonstrated that similar amounts of protein were added in each lane (Fig. 4).- K( ?3 b1 J4 b4 \2 o3 i$ M
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Figure 4. Western blot analysis. Protein extracts from 5-month-old rat brain culture (lane 1) and from day 0 (lane 2), day 5 (lane 3), day 10 (lane 4), and day 190 (lane 5) of HUCB culture. Actin was used as a loading control. Abbreviation: HUCB, human umbilical cord blood.

DISCUSSION
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Tanja Zigova died in February 2004, during the preparation of this manuscript. This paper is dedicated to the memory of Dr. Zigova: an erudite scholar, a visionary scientist, and a gentlewoman. She was a beloved mentor, an inspiration, and a friend. We are grateful to Marci McCall for editorial assistance. This work was supported by NIH/NIA grant R01 AG20927-01 (to T.Z.).
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DISCLOSURES0 B7 b9 I: M$ t2 @+ J

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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