Human Umbilical Cord Matrix Stem Cells: Preliminary Characterization and Effect - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:08 |只看该作者 |倒序浏览 |打印

作者：Mark L. Weissa, Satish Medicettya, Amber R. Bledsoea, Raja Shekar Rachakatlaa, Michael Choib, Shosh Merchavb, Yongquan Luoc, Mahendra S. Raoc, Gopalrao Velagaletid, Deryl Troyera作者单位：a Department of Anatomy and Physiology, Kansas State University, Manhattan, Kansas, USA; ' p' S: h# d9 e


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      【摘要】) K% P3 k" U- A% E5 V4 ?
   The umbilical cord contains an inexhaustible, noncontroversial source of stem cells for therapy. In the U.S., stem cells found in the umbilical cord are routinely placed into bio-hazardous waste after birth. Here, stem cells derived from human umbilical cord Wharton¡¯s Jelly, called umbilical cord matrix stem (UCMS) cells, are characterized. UCMS cells have several properties that make them of interest as a source of cells for therapeutic use. For example, they 1) can be isolated in large numbers, 2) are negative for CD34 and CD45, 3) grow robustly and can be frozen/thawed, 4) can be clonally expanded, and 5) can easily be engineered to express exogenous proteins. UCMS cells have genetic and surface markers of mesenchymal stem cells (positive for CD10, CD13, CD29, CD44, and CD90 and negative for CD14, CD33, CD56, CD31, CD34, CD45, and HLA-DR) and appear to be stable in terms of their surface marker expression in early passage (passages 4¨C8). Unlike traditional mesenchymal stem cells derived from adult bone marrow stromal cells, small populations of UCMS cells express endoglin (SH2, CD105) and CD49e at passage 8. UCMS cells express growth factors and angiogenic factors, suggesting that they may be used to treat neurodegenerative disease. To test the therapeutic value of UCMS cells, undifferentiated human UCMS cells were transplanted into the brains of hemiparkinsonian rats that were not immune-suppressed. UCMS cells ameliorated apomorphine-induced rotations in the pilot test. UCMS cells transplanted into normal rats did not produce brain tumors, rotational behavior, or a frank host immune rejection response. In summary, the umbilical cord matrix appears to be a rich, noncontroversial, and inexhaustible source of primitive mesenchymal stem cells.
      【关键词】 Whartons jelly Flow cytometry Regenerative medicine In vitro expansion Noncontroversial source of stem cells$ T- M: Z# k, |8 ?% N9 o
   INTRODUCTION
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Stem cells derived from embryos (embryonic stem cells .7 v, l& \, A# U1 L$ m

With regard to moral/ethical issues, postnatal stem cells offer fewer concerns. Dogma has it that, in contrast to fetus-derived stem cells or ESCs, postnatally derived stem cells have less broad developmental potential. This dogma has no real teeth: several labs have shown that postnatal stem cells have wide potential .1 U0 k% K6 O/ z; q. x

At least two types of stem cells have been found in umbilical cord/placental tissues: blood-forming stem cells (hematopoietic stem cells .8 u  p9 G/ h" a

The umbilical cord vessels and surrounding mesenchyme (including the connective tissue matrix that becomes Wharton¡¯s jelly) are derived from extraembyronic mesoderm and/or embryonic mesoderm. Thus, these tissues, as well as primordial germ cells, differentiate from the proximal epiblast around the time of primitive streak formation . Thus, these tissues are rich sources of stem cells that may be useful for a variety of therapeutic purposes. We speculate that the umbilical cord matrix material derives from primitive mesenchyme that is in a transition state to the mesenchyme found in the bone marrow niche of adult animals. Here, we investigate this hypothesis by characterizing human umbilical cord matrix stem (UCMS) cells and comparing them with stem cells derived from other sources., n! J  {3 ?- j! t: [) j# R

As mentioned above, umbilical cord tissues contain pluripotent stem cells. We speculated that these stem cells may be useful for treating neurodegenerative diseases, such as Parkinson¡¯s disease (PD). Previous work has indicated that cells derived from embryonic or fetal sources may be useful when transplanted into PD rodents . Here, to test the value of human UCMS cells, we expanded upon our previous work by testing the behavioral effects of human UCMS cells in the PD rat model.
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MATERIALS AND METHODS% ?$ j  o' {; i- a4 `

Human Subjects
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The work was conducted following approval of the Kansas State University human subjects board (IRB approval no. 3515).

Generation and Expansion of UCMS Cells
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The collection and expansion of UCMS cells was described previously . Briefly, human umbilical cords from both sexes were collected from full-term births (two sets of fraternal twins) with informed consent of the mother after either cesarean section (11 of 14) or normal vaginal delivery and aseptically stored at 4¡ãC in sterile saline until processing. To isolate UCMS cells, the cord was rinsed several times with sterile saline and cut into 2- to 4-cm lengths. The interval between collection and isolation of UCMS cells ranged up to 48 hours. To isolate UCMS cells, the cord blood was drained and clots flushed from the vessels. Next, the vessels were stripped manually from cord segments, the wall of the cord was opened and the tissue was immersed in an enzyme cocktail (e.g., hyaluronidase, trypsin, and collagenase) for 45 to 60 minutes at 37¡ãC. This tissue was then crushed with forceps to release individual UCMS cells, and large pieces of tissue were removed. The cells were pelleted by low-speed centrifugation (250g for 5 minutes), suspended in fresh medium, and plated onto hyaluronic acid (HA)¨Ccoated plastic plates. Pilot work indicated that HA-coated plates optimized the attachment of UCMS cells.

Several different media support the growth of adherent UCMS cells: a low serum, "defined media"¡ª56% low glucose Dulbecco¡¯s modified Eagle¡¯s media (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 37% MCBD 201 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 2% fetal bovine serum (FBS; MTT 35-010-CV; Mediatech, Herndon, VA, http://www.cellgro.com), 1x insulin-transferrin-selinium-A (ITS; Invitrogen), 1x ALBU-Max1 (Invitrogen), 1x Pen/Strep (Invitrogen), 1x Amphotericin-B (ICN-1672348, Fisher Scientific, Hampton, NH, http://www.fisherscientific.com), 10 nM dexamethasone (Sigma-Aldrich), 50 µM ascorbic acid 2-phosphate (Sigma-Aldrich), 1 ng/ml epidermal growth factor (EGF; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), 10 ng/ml platelet-derived growth factor-BB (PDGF-BB; R&D systems)¡ªor the same media without ascorbic acid and dexamethasone. This media was used for all the experiments described here. Cells were incubated at 37¡ãC in an incubator with 5% CO2 at saturating humidity. When cells reached 70%¨C80% confluency or when numerous colonies were observed, the cells were detached with 0.25% trypsin-EDTA (Invitrogen), the trypsin was inactivated with fresh media, and the cells were centrifuged at 250g for 5 minutes and replated on HA-coated plates 1:3. UCMS cells could be stored frozen at ¨C135¡ãC in a freezing media containing 1:1 defined media: 80% FBS, 20% dimethylsulfoxide, or in 93% FBS, 7% DMSO or 93% FBS, 7% glycerol, alone, after removal of the media. To demonstrate that human UCMS cells have the capacity for clonal expansion, clonal lines were established by the removal of a single cell to an individual well of a 24-well plate. The addition of a single cell per well was confirmed visually for each well using phase contrast microscopy. To determine whether human UCMS cells were karyotypically stable in culture, human UCMS cells from various passages .  j. h/ J% h8 L) K9 p

Total RNA

Total RNA was collected from human UCMS cells (various passages from 2¨C8). Human bone marrow MSCs were purchased from Cambrex (Walkersville, MD, http://www.cambrex.com) and grown using the manufacturer¡¯s protocol. Total RNA from MSCs was collected from passage 5. Undifferentiated human ESCs were obtained from WiCell Research Institute (H1; Madison, WI, http://www.wicell.org) using the supplied protocol. RNA was isolated either with TRIZOL LS (Invitrogen) using the manufacturer¡¯s protocol or by Qiagen RNeasy kit (Qiagen, Hilden, Germany, http://www1.qiagen.com) using manufacturer¡¯s protocol. The RNA was treated with DNase and stored at ¨C80¡ãC. Total RNA from universal somatic stem cell (USSC) and from nestin-positive islet precursors (NIPs) was supplied from ViaCell, Inc. (Cambridge, MA, http://www.viacellinc.com) (Andrei Kritsov and Elizabeth Abraham, respectively).
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Flow Cytometric Analysis of UCMS Cells

The cells were analyzed by flow cytometry in two independent labs (Kansas State University and ViaCell Singapore Research Centre). To stain the UCMS cells, the cells were lifted with trypsin and the trypsin was inactivated with fresh media. Approximately 106 cells were pelleted and resuspended in PBS and fixed with 4% buffered paraformaldehyde for 5 minutes at room temperature. For staining, the nonspecific binding was blocked with PBS with 2% normal serum for 5 minutes, then the cells were incubated with primary antibody for 45 minutes at room temperature. The cells were stained with a fluorescent secondary antibody for 30 minutes. Control cells were prepared by incubation with the secondary antibody alone. In each case, the cells were gently pelleted and washed with PBS rinses between each incubation step. For cytoplasmic antigens, the cells were permeablized by 100% cold methanol for 5 minutes. Antibodies used are shown in supplemental online Table 1.8 Q7 z" f0 L: W# @3 {+ i
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Reverse Transcription¨CPolymerase Chain Reaction

The cDNA was synthesized using 0.2 to 1 µg of total RNA in the presence of Superscript II and oligo(dT)12¨C18 (both from Invitrogen). The DNA sample was treated with RNase H and stored frozen until use. Polymerase chain reaction (PCR) was performed in a 20-µl reaction solution containing 2 µl of 10x PCR buffer, 150 µmol of MgCl2, 10 µmol of dNTP, 20 pmol of primer, 1 µl of 10x diluted cDNA, and 1 U of RedTag DNA polymerase (Sigma-Aldrich). The PCR conditions were as follows: 5 minutes at 94¡ãC followed by 35 cycles of 94¡ãC for 30 seconds, 55¡ãC for 30 seconds, and 72¡ãC for 60 seconds, and final extension for 10 minutes at 72¡ãC. Primer sequences are provided in supplemental online Table 2.! B; _3 T% \) [' [
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Focused Gene Array Analysis% Z* W4 D( K$ ^( F6 P, n- N

Nonradioactive GEArray cDNA expression array filters (HS-601.2; SuperArray Inc., Frederick, MD) were used, and hybridization procedures were as described by the manufacturer. Total RNA was checked spectrophotometrically or by gel. Biotin dUTP-labeled cDNA probes were specifically generated in the presence of a designed set of gene-specific primers using total RNA (4 µg per filter) and 200 U of Moloney Murine Leukemia Virus (MMLV) reverse transcriptase (Promega, Madison, WI, http://www.promega.com). The array filters were hybridized with biotin-labeled probes at 60¡ãC for 17 hours. The filters were then washed twice with 2x saline sodium citrate (SSC) buffer/1% SDS and then twice with 0.1x SSC/1% SDS at 60¡ãC for 15 minutes each. Chemiluminescent detection steps were performed by subsequent incubation of the filters with alkaline phosphatase-conjugated streptavidin and CDP-Star substrate (Roche Applied Science, Indianapolis, http://www.roche-applied-science.com). The microarray experiments were performed multiple times with new filters and with RNA isolated from different passages from the same umbilical cord and from different umbilical cords. The Z-transformed data from all samples are in supplemental online Table 3. Here, the averaged Z-transformed data from the 50 most highly expressed genes are presented (Table 1).
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Table 1. Fifty of the most highly expressed genes in human UMCS cells$ y( x3 u4 A) o  \! H

Testing the Therapeutic Effect of UCMS Cells in Parkinsonian Rats
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The Hemiparkinsonian Rat Model. Brain damage was induced by a single stereotaxic injection of 6-hydroxydopamine (6-OHDA) as previously described . Anesthetized female Sprague-Dawley rats received a 4.0-µl injection of 32 mM 6-OHDA HCl (Sigma-Aldrich) dissolved in 0.02% ascorbate at 1 µl/minute into the left medial forebrain bundle (Bregma ¨C2.8 mm, lateral  2.0 mm, ventral  8.4 mm, incisor bar 3.9 mm ventral to interaural axis) or an injection of vehicle (sterile saline) 4 weeks prior to transplantation. The lesioned animals showed spontaneous rotations upon recovery from lesion surgery. Following surgery, rats were monitored for behavioral changes, weight loss, and overall health. Four weeks following lesion surgery, the animals received the human UCMS cell transplant or a sham transplant (sterile saline). The efficacy of the 6-OHDA lesion was assessed by apomorphine-induced rotations every 2 weeks following the lesion and also by characterizing staining for tyrosine hydroxylase (TH) in the ipsilateral and contralateral striatum and ventral midbrain after sacrifice (sees below).' s5 E  x9 z, w9 @/ d

Behavioral Assessment. To test the efficacy of the lesion, rats were placed in round, opaque testing chambers and allowed to acclimate for 5 minutes prior to receiving the apomorphine injection (0.1 mg/kg s.c.). The number of rotations and the direction of rotation were noted for a 30-minute period after giving the apomorphine injection. Four weeks following the lesion, rats that did not exhibit at least 200 rotations/30 minutes toward the contralateral side after apomorphine treatment were excluded from the experiment. Recovery of function was evaluated over time (repeated measures) and pooled across groups to compare treatments.
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UCMS Transplantation. At 4 weeks after brain lesion, animals were behaviorally tested and randomly assigned to transplantation or sham-transplantation groups. Approximately 1000 human UCMS cells in 1 µl of sterile PBS were injected over 5 minutes into the striatum (Bregma  0.5 mm, lateral  3.4 mm, ventral ¨C5.0 mm from the surface of the brain). All animals received UCMS cells from the same passage (passage 9) on the same day. Before transplantation, the cells were lifted by a trypsin solution and were counted by a hemocytometer, and were adjusted to a final concentration of approximately 1000 cells/µl. The cell concentration was confirmed before and after the injection to ensure that approximately 1000 cells were delivered. Multiple control groups were used, including 1) lesioned rats with sham transplant (saline), 2) nonlesioned rats with human UCMS cell transplant, and 3) nonlesioned rats with sham transplant (saline). At 6 and 12 weeks post-transplant, 12 rats were randomly selected from the experimental groups, anesthetized, and sacrificed by transcardial perfusion with heparinized isotonic saline rinse followed by 10% buffered neutral formalin. The brains were removed, postfixed, and cryoprotected in 20% sucrose overnight. Frozen sections of the brains were cut coronally at 40 µm and the sections were collected into six sets of adjacent sections, each set consisting of every sixth serial section./ G" |, Q0 p" P8 Z: ?0 p$ N7 M

Tissue Processing and Immunocytochemistry

Immunocytochemical (IC) detection of a single antigen was performed on one set of sections as previously described . The free-floating tissue sections were stained with primary antibodies for anti-human nuclear antigen (mouse host, 1:30; Chemicon, Temecula, CA, http://www.chemicon.com), TH (rabbit host, 1:2000; East Acres Biologicals, Southbridge, MA), anti-rat CD4 (mouse host, 1:500; Serotec Ltd., Oxford, U.K., http://www.serotec.com), anti-rat CD8 (mouse host, 1:500; Serotec), anti-rat CD11b (mouse host, 1:250; Serotec), and anti-rat CD161 (mouse host, 1:250; Serotec). The antigens were visualized either with diaminobenzidine (DAB) and hydrogen peroxide using a commercially available ABC kit (Vectastain; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) or with immunofluorescence. For immunofluorescence localization, 7-amino-4-methylcoumarine-3-acetic acid (AMCA) (Avidin D; Vector Laboratories) was used with the biotinylated secondary antibody. The IC-stained sections were mounted on subbed microscope slides, air dried, and rinsed with distilled water. Immunofluorescence in the sections was observed using epifluorescence illumination with the appropriate filter combinations on a Leica DMRD microscope (Leica Microsystems Inc., Bannockburn, IL, http://www.leica-microsystems.com) after clearing and coverslipping with glycerol containing n-propyl gallate (three parts 2% n-propyl gallate in 0.1 M Tris buffer, pH 9.0, and seven parts glycerol).
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Counting TH-Positive Cells1 M4 ?+ _* _$ X' M  M
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The TH-positive dopaminergic (DA) neurons in the substantia nigra (SN) and ventral tegmental area (VTA) were counted using a design-based unbiased stereological method and a morphometry/image analysis system (Bioquant Nova Prime; R&M Biometrics; BIOQUANT Image Analysis Corporation, Nashville, TN, http://www.bioquant.com). One set of brain sections of all the experimental animals was stained with anti-TH antibody and visualized with DAB. In each section, the region of interest was outlined (SN and VTA) and the number of TH-positive cells/mm2 of that region was selected and semiautomatically counted. The SN and VTA TH-positive cells were counted, and the average number of TH-positive DA neurons/mm2 was calculated for each animal.2 R( S: H5 C2 s% p9 a3 S
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Statistical Analysis9 k1 R9 R2 s/ O3 v9 u

All tissue manipulations were conducted in large batches to avoid batch to batch variability in tissue IC staining. Data collection was conducted in an experimenter-blind fashion. Following data collection, the group status was decoded prior to statistical analysis. In general, analysis of variance (ANOVA) was used to evaluate group differences. ANOVA was followed by post hoc testing using Scheffe¡¯s test. Significance was set at p ( `. a" F8 C% c1 r

RESULTS
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Characterizing UCMS Cells9 n$ H6 R( n. i+ F& N  V8 [
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Culture. Human UCMS cells were isolated from 15 of 17 cords. We isolated 15 to 17 x 103 cells per cm of cord length (range, 10¨C50 x 103 cells/cm). The primary isolates were heterogeneous, with mesenchymal-like cells possessing short and long processes as well as small round cells with a high nuclear to cytoplasm ratio (supplemental online Fig. 1). Individual cells could be clonally expanded. Through 10 passages, the populations were morphologically and immunophenotypically similar to the parent cells (data not shown). Clonally derived lines of human UCMS cells were confirmed by visual inspection that a single UCMS cell was delivered per well of a 24-well plate. The clonal capacity was estimated at a rate of one to four clonal populations derived per 48 attempts (data not shown). The clonal lines were not analyzed further. Human UCMS cells from four isolates were GTG-banded and chromosomes were evaluated. None of the isolates were abnormal; the karyotype of the cells was stable through 13 passages (approximately 30 population doublings) (supplemental online Fig. 2)./ E6 d# Q' A9 J6 T2 j

Flow Cytometry and Sorting of Hoechst-Dim Cells. A relatively large percentage of UCMS cells exclude Hoechst dye (Hoechst-dim; average of 23%; n = 4; range, 8¨C32%). Approximately 85% of the Hoechst-dim-stained cells were also CD44-positive (Fig. 1). Interestingly, sorting Hoechst-dim cells did not enrich for CD44-stained cells (data not shown). To determine whether the Hoechst-dim/CD44-positive population could be enriched further and expanded, the Hoechst-dim population was sorted using fluorescence-activated cell sorting. The sorted Hoechst-dim staining cells appeared morphologically distinct from the Hoechst-bright population, with a higher percentage of the cells being small round cells (Fig. 1C, 1D). The enriched population of Hoechst-dim cells was expanded through five passages, and the percentage of Hoechst-dim and CD44-positive cells was again assessed. At this time, the two populations looked morphologically similar, and 23.5% of the cells were in the Hoechst-dim sorted population and 20% were in the Hoechst-bright population. Presently, enrichment for Hoechst-dim cells by flow sorting is not maintained after passage. Previous work has indicated that Hoechst-dim staining is a result of the presence of the ABCG2 receptor . As shown in Figure 1E and 1F, human UCMS (hUCMS) cells showed the characteristic surface staining for the ABCG2 receptor and CD44. Verapamil (VP; 10 µM) was used as a nonspecific blocker of the Hoechst dye exclusion channels. VP treatment partially blocked Hoechst dye exclusion, with 24% dye-excluding cells without VP and 7% dye-excluding cells with VP (data not shown).

Figure 1. Flow cytometry and fluorescence-activated cell sorting of human umbilical cord matrix stem (hUCMS) cells. (A): hUCMS cells stained for Hoechst 33342 revealed a population of cells showing low fluorescence (see box) that comprised approximately 20% of the cells. When this population was sorted out and stained for CD44 expression, approximately 85% of these Hoechst-dim cells expressed CD44 (B). The two populations, Hoechst-dim and Hoechst-bright, were expanded in vitro. The morphologies of the Hoechst-dim and Hoechst-bright populations are shown in (C) and (D), respectively. Following expansion and passage, the two populations were again Hoechst- and CD44-stained. Immunocytochemistry was used to verify ABCG2, the transporter thought to be responsible for Hoechst dye efflux (E), and CD44, a mesenchymal cell marker that is the hyaluronate receptor (F)./ c3 o- d5 K% v6 I

Flow Cytometry. Human UCMS cells from passage 4 (P4) and passage 8 (P8) from two different cord samples were characterized by flow cytometry (Table 2; representative histograms of these data can be found in online supplemental Fig. 3). UCMS cells stained positively for CD10 (average of 96% in P4 and 86% in P8), CD13 (average of 91% in P4 and 94% in P8), CD29 (average of 98% in P4 and 98% in P8), CD44 (average of 96% in P4 and 94% in P8), CD90 (average of 91% in P4 and 67% in P8), and HLA-1 (average of 86% in P4 and 79% in P8). In later passages, a smaller number of UCMS cells expressed CD49e (average of 83% in P4 and 16% in P8) and CD105 (average of 86% in P4 and 24% in P8). CD14, CD31, CD33, CD34, CD45, CD56, CD133, and HLA-DR were not detected (Table 2). Note that the percentage of cells staining was fairly stable over four passages examined with no clear trends (Table 2).$ h* P8 q& B4 p7 m0 b
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Table 2. Flow cytometry data9 k0 K4 A! m& V
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Neural Potential of hUCMS Cells. The majority of undifferentiated UCMS cells stained for glial cell line-derived neurotrophic factor (GDNF; Fig. 2A). Immunocytochemistry for GDNF showed the staining to be evenly distributed throughout the cytoplasm of the cells (Fig. 2B). Neural induction, using the methods described by Woodbury et al. , resulted in a lower number of cells expressing the marker for early neural progenitors (i.e., nestin) and a greater number of cells expressing the marker for catecholaminergic cells, TH, a mature neural marker (Fig. 2C, 2D).

Figure 2. Neural potential of human umbilical cord matrix stem (hUCMS) cells. Production of GDNF by hUCMS cells. More than 80% of the cells were positive for GDNF as revealed by flow cytometry (A). Immunocytochemical staining confirmed the flow cytometry findings (B). (C, D): Effect of the differentiation of UCMS cells on the expression of nestin and TH staining. The expression of nestin, a marker of primitive neural stem cells, was lower in differentiated cells (10.03%) than in undifferentiated cells (23.41%) (C). The expression of TH, a marker of catecholaminergic neurons, was greater in differentiated cells (63.23%) than in undifferentiated cells (20.1%) (D). Abbreviations: GDNF, glial cell line-derived neurotrophic factor; TH, tyrosine hydroxylase.+ }5 Q# b" W; z; x3 }" f6 a

Gene Array Results for Undifferentiated hUCMS Cells. hUCMS cells express many different genes, suggesting that they are promiscuous in the production of genes, as has been described for other stem cells such as HSCs and ESCs . The 50 genes that were most highly expressed by hUCMS cells are shown in Table 1. (The Z-transformed data for all array elements are provided in supplemental online Table 1). Of the top 50 genes, genes found expressed in undifferentiated ESCs that were also expressed in the UCMS cells were leukemia inhibitory factor receptor (LIFR) pathway, ESG1, SOX-2, and TERT. hUCMS cells expressed genes for proteins associated with morphogenesis: SHH, neuregulin-1 and 4, Patched, SNA2, and WNT4. In addition, hUCMS cells expressed extracellular adhesion molecules: N-cadherin, V-cadherin, R-cadherin, integrin-ß1, integrin-5, VCAM-1, integrin-2 (CD49b), integrin-V, integrin-ß5, integrin-4, and integrin-3. hUCMS cells expressed genes of proteins shown to have a neurotrophic effect: CNTF, VEGF, FGF20, and TRKC. hUCMS cells expressed markers of the three germ layer derivatives: mesoderm, ACTG2, ACTA2, BMP1, PDGFB; ectoderm, keratin 8, SHH; endoderm, insulin. In contrast to what was seen with the gene array, neither flow cytometry (Table 2) nor RT-PCR (data not shown) detected CD34.! N7 M% b2 m. c" N

RT-PCR. The gene-expression profile of UCMS cells and two other cell types¡ªNIP cells (a generous gift from Elizabeth Abraham, ViaCell, Inc.) and bone marrow-derived MSCs¡ªand positive human control RNA (liver, placenta, and muscle; BioChain, Hayward, CA, http://www.biochain.com) was evaluated using RT-PCR to confirm or expand the gene array results. As shown in Figure 3A, hUCMS cells expressed markers of the undifferentiated state, Oct4, FGFR4, LIFR, Glut-1, ABCG2, Nanog, and Rex-1 as detected by RT-PCR. hUCMS cells also expressed genes of proteins from all three germ layers and trophectoderm. Trophectoderm markers were Bex1/Rex3, Hand1, and HEB (Fig. 3B). The neurectoderm marker was nestin; mesoderm markers were CXCR4, vimentin, CD44, collagen X, and Flk-1 (Fig. 3B, 3C); and endoderm markers were PDX1 and islet-1. Figure 3C shows the expression of UCMS cells compared with that of MSCs. The two cell types were similar in the expression of OPN, ABCG2, CD44, LIFR, and collagen I, 1. One notable difference was that the UCMS cells expressed Flk-1.8 h0 S. b" \; V' B

Figure 3. Reverse transcription¨Cpolymerase chain reaction data comparing human umbilical cord matrix stem (hUCMS) cells with various other cell types. Cells were compared using markers for pluripotent cells (A), trophectoderm or germ layers (B), and other miscellaneous markers (C). NIPs were supplied by ViaCell, Inc.; bone marrow¨Cderived MSCs were from Cambrex; and ESC indicates a mixed total RNA from several approved human embryonic stem cell lines (supplied by MSR). Control RNA was from placenta (top three rows), liver (rows 4¨C8), and heart muscle (bottom row). Abbreviations: NIP, nestin-positive islet precursor; MSC, mesenchymal stem cell; ESC, embryonic stem cell.
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Effect of UCMS Cells in Parkinsonian Rats

Behavioral Assessment. The PD model rats that received a human UCMS cell transplant showed a significant decrease in the number of rotations that was not seen in sham-transplanted animals (Fig. 4). Non-PD model animals with and without an hUCMS cell transplant did not rotate following apomorphine treatment (these data points overlay each other in Fig. 4A). In retrospect, we concluded that the grafts were unlikely to be effective in all animals, so post hoc, the animals that responded to the transplant, that is, decreased their rotations following transplant of hUCMS cells, were identified (indicated by boxed region in Fig. 4B). The data from individual animals are shown Figure 4B. These data show that at 6 weeks post-transplant two of four animals responded (decreased rotations by approximately 60%). At 12 weeks¡¯ survival, three of four animals responded to the transplant (decreased rotations by approximately 60%). One animal in the 12-week survival group decreased rotations by 90%. In contrast, in this experiment, the PD animals that were not transplanted with UCMS cells all showed a progressive degradation in performance following 6-OHDA. Behavior of the nonresponder animals and the control animals was not significantly different.7 O9 a" l' l0 e+ [

Figure 4. Effect of human umbilical cord matrix stem (hUCMS) cells on Parkinson¡¯s disease (PD). (A): Apomorphine-induced rotations (percentage change after transplantation surgery) in animals that received hUCMS cell or sham transplants. The number of rotations made at the time of transplantation is taken as 100%. The number of rotations observed in the PD model rats (6-OHDA) that received an hUCMS cell transplant was significantly lower than the control PD rats that received a saline injection. The lesion control rats (no lesion), both with hUCMS cell transplants and saline injection, did not show rotational behavior. (B): Inspection of the rotational behavior of individual PD model rats that received hUCMS cell transplants revealed two populations: one population decreased their rotations (responders, indicated in box); the other population did not. Note that at 6 weeks, two of four rats responded, and at 12 weeks, three of four rats responded to the hUCMS cell transplant. Abbreviation: 6-OHDA, 6-hydroxydopamine.

Histological Assessment of 6-OHDA Lesion. Unilateral staining of TH in the DA neurons of SN and VTA on the contralateral side of the lesion showed evidence of the toxic effect of 6-OHDA on DA neurons on the ipsilateral side of the injection site (Fig. 5A). The loss of DA neurons was restricted to the SN and VTA. In addition, unilateral TH staining was observed in the striatum, indicating that the ipsilateral nigrostriatal tract was destroyed by 6-OHDA (not shown). Figure 5A shows the TH staining from the experimental groups at the short and long survival time points. In the PD model rats 6 weeks after human UCMS cell transplantation, there was no TH-positive staining in the ipsilateral SN and VTA, and a slight degree of TH immunoreactivity was observed at 12 weeks after transplantation (Fig. 5A, bottom left). The PD model rats that received a sham transplant did not show any changes in TH staining at the longer survival time point. In intact, non-PD model rats with or without UCMS cell transplants, TH staining was observed in the SN and VTA at both survival time points, and no enhanced TH staining was observed around the transplant site (data not shown).
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Figure 5. Effect of transplant on TH staining. (A): Immunocytochemical staining for TH in the ipsilateral (left) and contralateral (right) SN and VTA of transplanted and control animals at the 6-week (top) and 12-week (bottom) survival time points. (B): Effect of hUCMS cell transplant on the number of TH-positive cells in the midbrain of Parkinson¡¯s disease model rats. In responder animals (animals demonstrating behavioral recovery following hUCMS cell transplant), but not in control animals or in nonresponder animals, more TH-positive cells were found in the ipsilateral (left) and contralateral (right) side 12 weeks following transplantation. The largest effect was seen in the SN, but increases were also seen in the VTA (data not shown). Abbreviations: TH, tyrosine hydroxylase; SN, substantia nigra; VTA, ventral tegmental area; hUCMS, human umbilical cord matrix stem.  _/ a3 N/ Q5 o; e* o
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Counting TH-Stained Cells. We quantified the number of TH-positive cells in the SN and VTA; these results are shown in Figure 5B. There was a correlation with the behavioral recovery at 12 weeks, but not at 6 weeks following surgery. At 6 weeks, there was no effect of the transplant on the number of TH-staining cells in the SN or VTA on either the ipsilateral or contralateral side (Fig. 5B). In contrast, at 12 weeks¡¯ survival, the animals that responded to the transplant had significantly more TH-stained cells in the SN and VTA ipsilateral and contralateral to the transplant. In nonresponder animals, there was no difference in the number of TH-stained cells compared with the sham-transplanted controls.% y- |, P* }6 n0 d! n

Recovery of the Transplanted Cells. hUCMS cells were recovered 2 days following transplantation by staining the brain sections with anti-human nuclear (AHN) antigen (supplemental online Fig. 4, left). However, no positive-staining human cells were found at 6 weeks or 12 weeks following UCMS cell transplant.( h+ \# b9 x$ ]: |$ r7 i, @& O

Immune Cell Infiltration. One set of brain sections from selected animals of the 6-week and 12-week survival groups was stained with anti-CD8 antibody (recognizes T-cytotoxic cells) and visualized by AMCA. Brightly stained CD8 cells were seen at the lesion site (supplemental online Fig. 4, right), but not at the transplantation site. Similar staining was observed with the CD4 (recognizes T-helper cells) and CD11b (recognizes activated microglial cells) antibodies.
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DISCUSSION
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Here, hUCMS cells were characterized in vitro and tested for their therapeutic potential in a PD rodent model. Three novel observations were made. First, UCMS cells had surface markers that indicate that they maybe a primitive MSC population. Second, UCMS cells expressed genes found in early development and genes associated with the three principal germinal layers, that is, ectoderm, mesoderm, and endoderm. Third, UCMS cells could partially reverse the parkinsonian behavioral phenotype in rats. Taken together, these results suggest that the umbilical cord matrix may be an important, noncontroversial source of primitive stem cells that may be harvested at low cost and in large numbers for cryogenic banking.
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During development, primordial germ cells and hematopoietic cells migrate from the secondary yolk sac through the umbilical region into the embryo proper. Thus, the cell described here may be germ or hematopoietic-type cell that is retained in the hyaluronic acid-rich extracellular matrix. Alternatively, the cell described here may be a support cell (stroma cell) like those found adjacent to other stem cells (as is in the bone marrow cavity, intestinal crypts, etc.). It is important to note that other labs have isolated stem cells from various umbilical or placental structures as well as the amniotic fluid. Clearly, these primitive structures, like UCB, are important, noncontroversial sources of stem cells. From the characterization of the cells derived from other sources, for example, the umbilical vein . Further work is needed to confirm this hypothesis.

Properties of hUCMS Cells+ M; \; ?+ M& J- v6 k7 J

Previously we reported that UCMS cells could be started via explant culture of the minced cord matrix. Here, we report a more efficient method of starting the cultures via enzymatic degradation of the extracellular matrix to release the cells from the Wharton¡¯s jelly. UCMS cells were isolated from 78% of the cords, including two cords that were refrigerated for 24 hours prior to isolation (cells were isolated from one of the two refrigerated cords). Since this improved procedure has been used, the isolation of UCMS cells has been 100%. Many cells are found in the initial extraction from the cord, and this provides the opportunity to sort out special populations early on. Adherent cells were obtained, expanded, and passaged, and could be frozen, thawed, and cultured. The attached UCMS cells had a fairly uniform morphology that is similar to that of MSCs isolated from bone marrow. Flow cytometry revealed that 20%¨C30% of early passage UCMS cells excluded Hoechst dye. The Hoechst dye exclusion was lower after treatment with VP, an inhibitor of some members of the ABCG protein family. Flow sorting of Hoechst-dim cells (also called side population . Thus, cord matrix has the highest percentage of SP cells known (range, 8¨C32%). Flow cytometry of the UCMS cells revealed that approximately 85% of UCMS cells stain for CD44, the HA receptor. CD44 is a marker found in other stem cell populations including MSCs and neural stem cells.6 U( ?  [$ }3 s3 ^" x

Flow Cytometric Analysis Indicates That UCMS Cells Have MSC Markers0 z( C5 b2 y: b9 A$ M6 S# {2 [

As reported by Pittenger et al. .

RT-PCR Results
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UCMS cells are pleiotropic, expressing many genes, such as those found in ESCs and other primitive stem cells, and genetic markers of all three germ layers, ectoderm, endoderm, and mesoderm. This finding is similar to what has been reported for other primitive stem cells and suggests that gene silencing or DNA methylation maybe important mechanisms to regulate UCMS differentiation. Currently, we have no data to address the mechanisms regulating UCMS differentiation.. {2 L! D" Y, V1 a8 N9 |

Gene Array Results

Of the 50 most highly expressed genes, the genes expressed by UCMS cells fall roughly into several categories: those associated with the undifferentiated state of ESCs, extracellular adhesion molecules, those associated with neurotrophic effects and morphogenesis, and those associated with the three germ layers. MSCs derived from UCB have recently been analyzed using microarray analysis and were found to express genes often found in MSCs derived from other sources . Technical differences such as the media used to expand the cells, the length of time in culture, and differences in gene array methodologies prevent the comparison of extant data sets. Such difficulties have been noted previously. To our thinking, comparisons of gene array data are best made from data collected within a single laboratory until such time as there is agreement on standardized media, sera, growth conditions, etc.
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Transplantation of UCMS into PD Rats$ @2 t- s6 ^8 Q5 j3 a

As widely reported previously, unilateral injection of 6-OHDA into the medial forebrain bundle caused destruction of the nigrostriatal DA neurons in the ipsilateral SN and VTA. This destruction produced motor abnormalities analogous to those seen in PD. The hemiparkinsonian rats had motor deficits indicated by rotational behavior following apomorphine injection. The rats had to meet a criterion of 200 apomorphine-induced rotations per 30 minutes for inclusion in our experiment. The groups were randomly divided into those that received a transplantation of hUCMS cells and a control group. Approximately 1000 hUCMS cells were transplanted into PD model rats in a single day from a single aliquot of freshly prepared cells. None of the animals received immunosuppressive therapy because our preliminary work in which we transplanted pig UCMS cells showed that the transplanted cells were not rejected . Moreover, pig UCMS cells survived and proliferated for up to 8 weeks after transplantation.! s! U8 W$ \$ t* x' k3 h% L

Here, the transplanted animals showed a significant recovery as a group. As individuals, when the data were inspected post hoc, it was revealed that >50% of the transplanted animals demonstrated behavioral recovery of approximately a 50% reduction in the rotational behavior. One of the animals showed a greater reduction in rotational behavior. Following sacrifice, IC for TH revealed no changes in the number of cells in the SN or VTA at the 6-week survival time point¡ªthis is despite the fact that behavior recovery was observed and accounting for the animals that responded to the transplantation. In contrast, in the responding transplanted animals that survived 12 weeks, there was a significantly greater number of TH-stained cells recovered in the SN and VTA. Most interestingly, the number of TH-stained cells was greater ipsilateral and contralateral to the lesioned nigrostriatal pathway. The mechanism that underlies this observation is unknown. Hypothetically, a rescue of dying nigrostriatal TH neurons may be stimulated by growth and neurotrophic factors released by the UCMS cell transplant. The hUCMS cells produce significant amounts of GDNF (Fig. 4), one of the most potent trophic factors for DA neurons, and fibroblast growth factor 20 (supplemental online Table 3), which is thought to be an important survival factor for DA neurons that is preferentially expressed in rat brain SN . On the other hand, UCMS cells may have properties similar to those of bone marrow stromal MSCs of inhibiting the host immune response. Dampening the host immune response may limit secondary damage and permit moribund neurons to recover.$ m0 r: y) F" a4 y
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We could not recover the transplanted hUCMS cells. Possible reasons for this include 1) the graft cells are cleared by the host immune system and 2) the low-density graft was not able to support itself and the cells could not survive for a long time. Because of the fact that we found immune cells at the lesion site, but not the transplantation site, it is not clear if the transplanted cells were scavenged by immune cells. However, there is a possibility that the transplanted cells were cleared and the immune cells left the area, as previous work with fetal pig mes-encephalic tissue grafted into the rat brain indicates that the grafted tissue is rejected in approximately 4¨C6 weeks . We have no direct or indirect evidence for a host immune response to the transplanted cells.; ?/ x! l( ^/ U* \4 _
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That the low-density graft was not recovered may also be a result of death of the grafted cells. Again, we have no direct evidence that the graft cells died, for example, there was no cellular debris, increased astrocyte infiltration, etc., that would indicate that the graft cells died. That we can recover human graft cells for up to 1 week following transplantation and recover pig umbilical cord matrix cell micrografts up to 8 weeks following transplantation into rats  suggests that our transplantation protocols are responsible for the problems recovering the grafted cells.# p8 m- k3 X0 ~2 M' X( x
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We observed a significant decrease in apomorphine-induced rotations in the PD model rats. There was a significant decrease in the number of rotations 4 weeks following transplantation that continued for up to 12 weeks after transplantation. Irrespective of the survival period, out of eight PD model animals that were transplanted with hUCMS cells, four showed a 40%¨C50% decrease in the number of rotations, one animal showed a 90% decrease, and three did not respond. The three animals that did not respond to the transplantation therapy showed a slight increase in the number of rotations, which can be attributed to the clearance of the graft cells earlier than in the others. The TH-positive DA neurons in the SN and VTA showed a valid correlation between the number of cells and the number of apomorphine-induced rotations, that is, the higher the number of TH-positive cells in the SN and VTA, the lower the number of rotations. Therefore, the behavioral recovery of the PD model animals could be a result of rescue of the degenerating DA neurons in the SN and VTA, which could be mediated by various trophic factors . Further analysis is necessary to prove this hypothesis. The above data from UCMS cells indicate that these cells may be therapeutically useful in treating central nervous system disorders.
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DISCLOSURES- n; r: U* Y- w: g/ {- H
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS( l6 z) j& E) L& X
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The characterization work was conducted at the National Institute on Aging (NIA) during my sabbatical leave; thank you to my host laboratory: Dr. M.S. Rao and the members of the Stem Cell Laboratory at NIA. Thanks to my wife, Betti G. Weiss, and my children, Rita, Jonathan, Ellen, and James, for their patience and understanding during my absence. Dr. S. Bennet is thanked for assisting with umbilical cord collection. Thanks to the Calvello family (Ed, Emilie, and Stefano) for housing me in Baltimore. The anonymous donors are thanked for donating umbilical cords. K. Becker, M. Pyle, J. Hix, R. Rakasheklar, D. Davis, R. Carlin, J. Cai, Y. Lui, T. Miura, H. Xue, J. Osborn, M. Mughal, T. Cocksaygan, S. Saxena, and I. Ginis are thanked for their assistance in this work. Our collaborators at ViaCell, Inc., E. Abraham and A. Krivtsov, M. Kraus, and J. Visser are thanked for donating total RNA from NIPs and USSC. This work was supported by the NIH (salary support during sabbatical leave), the Kansas State University (KSU) Department of Anatomy and Physiology, the KSU College of Veterinary Medicine Dean¡¯s Office, Terry C. Johnson Center for Basic Cancer Research, and NIH NS034160. M.L.W. and S.M. contributed equally to this work./ O( I& s( s* g1 k, ~; a5 @
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