 |
Introduction
|
---|
Somatic human stem cells which can be propagated in large quantities while retaining their ability to differentiate into different tissue cell types could serve as a highly valuable resource for the development of cellular therapeutics (1). Although embryonic stem cells have the broadest differentiation potential (2), their use for cellular therapeutics is excluded for several reasons: the uncontrollable development of teratomas in a syngeneic transplantation model (3), imprinting-related developmental abnormalities (4), and ethical issues (1). On the other hand, previously published claims that adult tissuespecific stem cells possess an intrinsic differentiation potential to other tissues may have been premature. Over the past years, studies have shown that after hematopoietic allo-transplantation with BM or G-CSFmobilized blood, a small percentage of donor cells can also be detected as nonhematopoietic tissue (59). However, this so-called "plasticity" appears to be an extremely rare event and may not carry a physiologically relevant impact in vivo (10). Moreover, subsequent data have shown that certain results may be the result of cell fusion (11). Recently, a rare cell from BM of rodents, called multipotent adult progenitor cell (MAPC), was identified which differentiated in vitro into cells of all three germ layers and contributed to most somatic tissues when injected into an early murine blastocyst (12). A phenotypically identical cell was isolated from human BM (13). It is unclear, however, whether such MAPCs decline with donor age, a phenomenon that has been observed for the hematopoietic (14) and the mesenchymal stem cell (MSC) compartment from BM (15). In contrast to adult BM, the stem cell compartment in cord blood (CB) is less mature. This has been documented for the hematopoietic stem cells, which in CB are more abundant than in BM and have a higher proliferative potential associated with an extended life span and longer telomeres (1619). Besides this biological superiority, CB is abundantly available, is routinely harvested without risk to the donor, and infectious agents such as CMV are rare exceptions (20)a definite advantage for the development of cell therapeutics in regenerative medicine. We identified a rare, CD45 and HLA class IInegative stem cell candidate displaying robust in vitro proliferative capacity without spontaneous differentiation but with intrinsic and directable potential to develop into mesodermal, endodermal, and ectodermal cell fates. Thus, we termed the primary population unrestricted somatic stem cell (USSC).
 |
Materials and Methods
|
---|
Generation and Expansion of USSCs.
CB was collected from the umbilical cord vein with informed consent of the mother (21). USSCs were generated from 94 (40.3%) of 233 CB. The mononuclear cell fraction was obtained by Ficoll (Biochrom) gradient separation followed by ammonium chloride lysis of RBCs. Cells were plated out at 57 x 106 cells/ml in T25 culture flasks (Costar). Two different media were used to initiate growth of the adherent USSC colonies: myelocult medium (StemCell Technologies) and low glucose DMEM (Cambrex) with 30% FCS, dexamethasone (107 M; Sigma-Aldrich), penicillin (100 U/ml; Grünenthal), streptomycin (0.1 mg/ml; Hefa-pharma), and ultraglutamine (2 mM; Cambrex). Expansion of the cells was performed in the same media but with a lower concentration or in the absence of dexamethasone. Cells were incubated at 37°C in 5% CO2 in a humidified atmosphere. When cells reached 80% confluency, they were detached with 0.25% trypsin (Cambrex) and replated 1:3 as outlined in Table S1 available at http://www.jem.org/cgi/content/full/jem.20040440/DC1. USSC karyotyping was performed by cytogenetic standard protocols.
Monoclonal Antibodies for Immunophenotyping of USSCs.
FITC-conjugated Ab CD4 (13B8.2), CD8 (B9.11), CD11a (25.3.1), CD14 (RMO52), CD15 (80h5), CD31 (5.6E), CD33 (D3HL60), CD34 (581), CD44 (J-173), CD62L (DREG56), CD80 (MAB104), CD90 (F15.42), CD71 (YDJ122), HLA-ABC (B9.12.1), F(ab')2 goat antimouse IgM/IgG, and isotype controls were from Beckman Coulter; CD45 (2D1), CD49b (AK7), CD86 (FUN-1), and CD40 (5C3) were from BD Biosciences; CD62E/P (1.2B6) were from Serotec; CD105 (SN6) was from Caltag; and CD106 (BBIG-V3) was from R&D Systems. PE-conjugated Ab CD11b (BEAR1), CD16 (3G8), CD25 (B1.49.9), CD29 (K20), CD38 (T16), CD49e (SAM1), CD50 (HP2/19), CD54 (84H10), CD56 (NKH-1), CD117 (95C3), and isotype controls were from Coulter; glycophorin A (GA-R2), CD10 (HI10a), CD13 (L138), CD49d (L25.3), CD49f (GoH3), CD73 (AD2), CD123 (9F5), CD166 (3A6), and HLA -DR (G46-6) were from BD Biosciences; and CD133/1 (AC133) and CD133/2 (AC141) were from Miltenyi Biotech. Unconjugated Ab CD58 (AICD58) and isotype controls from were Immunotech; FLK-1/KDR (A-3), cytokeratin 8 (C51), cytokeratin 18 (DC-10), CK8/18 (C51), and vimentin (V9) were from Santa Cruz; CD44H (2C5) was from R&D Systems; and human endo (P1H12) was from Chemicon. Goat antirat Cy2 and normal rat IgG were from Dianova. Analysis was performed on a Beckman Coulter EPICS XL-MCL.
RT-PCR Analysis of USSCs.
RT reactions were performed at 50°C with Omniscript (QIAGEN) and the 3' primers following supplier's instruction. The PCR reactions were performed with HotStar Taq Master Mix (QIAGEN) at 93°C for 5 min, 35 x 93°C for 30 s, 55°C for 30 s, 72°C for 1 min, and 72°C for 5 min. Expression of the following molecules was detected by RT-PCR: CD49e (5'-ggcttcaacttagacgcgg; 3'-ccaggttgatcaggtactc; 640 bp), CD105 (5'-cctgccactggacacagg; 3'-atggcagctctgtggtgttg; 411 bp), CHAD (5'-aggaaccagctgtccagct; 3'-agtcaccaggactggctg; 513 bp), PDGFRa (5'-acagtggagattacgaatgtg; 3'-cac-atcagtggtgatctcag; 251 bp), EGFR (5'-tgccacaaccagtgtgct; 3'-gaccagttcatcagattcatc; 205 bp), IGFR (5'-cgagtggagaaatctgcgg; 3'-gaccagggcgtagttgtag; 272 bp), RUNX1 (5'-gcaagctgaggagcggcg; 3'-gaccgacaaacctgaggtc; 296 bp), and GAPDH (5'-ctcaagatcatcagcaatgcc; 3'-gatggtacatgacaaggtgc; 755 bp).
Telomere Length Measurement.
Telomere length measurement was performed with the Telo TAGGG Telomere Length Assay (Roche Diagnostics) according to manufacturer's instructions. DNA was treated with RsaI and Hinfl. Detection was performed with Image Master (Amersham Biosciences). Calculation of mean telomere restriction fragment lengths (mean TRF) was performed according to Harley et al. (22).
In Vitro Differentiation into Neural Cells.
USSCs were seeded on glass coverslips coated with 1 mg/ml poly-D-lysine and 13 µg/ml laminin in differentiation medium XXL containing DMEM, 15% heat-inactivated FCS, 100 U/ml penicillin/streptomycin, 50 ng/ml nerve growth factor, 20 ng/ml bFGF, 1 mM dibutyryl cAMP, 0.5 mM isobuthyl methyl xanthine (IBMX), and 10 µM retinoic acid for up to 4 wk. For immunostaining, cells were fixed for 15 min with 4% paraformaldehyde (PFA). For immunostaining against gamma amino butyric acid (GABA) and tyrosine-hydroxylase (TH), cells were fixed in 4% PFA, 0.3% glutaraldehyde at RT for 5 min. After washing with PBS, cells were incubated in 1 M ethanolamine at RT for 20 min, preincubated and permeabilized in 10% normal goat serum with 0.03% Triton X-100 for at least 30 min, followed by incubation with the primary Ab (GABA 1:1000 (Sigma-Aldrich), TH 1:100 (Sigma-Aldrich), neurofilament (NF) cocktail 1:1,000 (BioTrend), DOPA-decarboxylase 1:500 (Sigma-Aldrich), glial fibrillary acidic protein (GFAP) 1:300 (Chemicon), synaptophysin 1:100 (Sigma-Aldrich), and Na+ channel 1:50 (Sigma-Aldrich). Secondary Ab were antimouse-FITC (1:100; Southern Biotechnology) and antirabbit rhodamine X Ab (1:1,000; Molecular Probes). Cell nuclei were labeled with 4-6'diamidino-2-phenylindoline (DAPI; Roche).
USSC Transplantation into the Hippocampus Region of Wistar Rats.
USSCs were labeled with pKH26 (Sigma-Aldrich) according to the supplier's protocol 2 d before transplantation. Adult male Wistar rats were anesthetized with Rompun (5 mg/kg) (Bayer) and Ketavet (100 mg/kg) (Pharmacia Upjohn). For transplantation, animals received a single, unilateral stereotactic injection of 1 µl (75,000 USSC/µl) into the hippocampus region using a 22-gauge needle. Coordinates were set according to the atlas of Paxinos and Watson (23). The animals received immune suppression by s.c. injection of cyclosporin A (15 mg/kg Sandimmune; Novartis) for 10 d, starting with a double dose injection 1 d before surgery. At different time points after transplantation the animals were killed. The brains were immediately removed and frozen in methyl butane (Sigma-Aldrich) on dry ice. 20-µm coronal sections were cut through the brain using a freezing microtome (CM 3050; Leica). At intervals of
400 µm, sections were stained for human Tau protein (hTau). Sections were fixed with 4% PFA for 10 min followed by 50%, 100%, 50% acetone (Merck), each for 2 min. After a washing step, sections were quenched by 0.3% H2O2 (Merck) for 30 min and blocked with 3% goat serum for 30 min. The hTau Ab (1:100; Chemicon) was incubated overnight at 4°C. After washes with PBS, sections were incubated with goat antirabbit biotinylated secondary Ab (1:200; Vector) for 1 h and rinsed with PBS. Later, sections were transferred to an avidinbiotin complex Vectastain ABC kit (Vector) and were developed with 3,3-diaminobenzidine (DAB; Sigma-Aldrich) and H2O2 for 10 min, dehydrated, and mounted in Entellan (Merck).
In Vitro Differentiation into Osteoblasts, Chondroblasts, and Adipocytes.
For differentiation into osteoblasts, USSCs were plated at 8,000 cells/cm2 in 24-well plates and at 70% confluency supplemented with 107 M dexamethasone, 50 µM ascorbic acid-2 phosphate, and 10 mM dexomethasone, asorbic acid, ß-glycerol phosphate (DAG) (24). For Alizarin red staining, cells were fixed for 5 min with 70% ethanol at 4°C to determine calcium deposition. Alkaline phosphatase (ALP) activity was determined in cell lysates obtained by treating cell cultures with 1% Triton X-100. The ELISA-based method was performed according to the manufacturer's protocol (Sigma-Aldrich). Results are expressed in nmol of p-nitrophenol produced per min. For quantitative Ca2+ determination, cell layers were scraped off the dish in 0.5 N HCl according to manufacturer's instructions (Sigma-Aldrich). For chondrogenic differentiation, a micromass culture system was used (25). 2 x 105 USSCs were cultured in DMEM high glucose supplemented with antibiotic, 100 nM dexamethasone, 35 µg/ml ascorbic acid-2-phosphate, 1 mM sodium pyruvate, ITS + premix (1:100 dilution), and 10 ng/ml TGFß1. Aliquots of 2 x 105 cells in 0.5 ml of medium were centrifuged at 150 g in 15 ml polypropylene conical tubes. The pelleted cells were incubated at 37°C and 5% CO2 for 21 d. For Alcian blue staining, cell aggregates were fixed in 4% formalin and cut into 10-µm sections and stained for histology. For immunostaining, frozen sections were fixed with 100% ethanol, incubated in 0.2 U/ml chondrotinase ABC for 40 min at 37°C. Blocking of nonspecific Ab binding sites was performed in 5% BSA/PBS for 1 h. The sections were incubated with the primary Ab diluted in 0.5% BSA/PBS for 1 h. Collagen type II (Chemicon) was detected by fluorescence microscopy after incubation for 30 min with a FITC-labeled secondary Ab diluted in 0.5% BSA/PBS. To induce differentiation into adipocytes, cells were plated at 1,000 cells/cm2 in 24-well plates in DMEM with 1 µM dexamethasone (Sigma-Aldrich), 10 µg/ml insulin, 0.5 mM IBMX (Sigma-Aldrich), and 100 µM indomethacin (Sigma-Aldrich) (26). After 2 wk of adipogenic stimulation, cells were fixed in 5% PFA for 30 min and incubated with Oil Red-O to stain lipid vacuoles.
In Vivo Differentiation into Bone and Cartilage.
The surgical details of the femoral gap bone repair model were performed according to a method described by Bruder et al. (27), and differentiation into cartilage was shown previously (28).
In Vitro Differentiation into Hematopoietic Cells.
105 USSCs were expanded for 2 wk with 100 ng/ml Flt3-L (CellGenix), 100 ng/ml SCF (CellGenix), 100 ng/ml IL-3 (Cellsystems), 100 ng/ml IL-6 (CellSystems), 100 ng/ml TPO (CellGenix), and 100 ng/ml G-CSF (Amgen) in Myelocult medium as described previously (29). Human CFU assays were performed on days 0 and 14 (29) with 104 cells in Methocult (Stem Cell Technologies).
Detection of In Vivo Differentiation into Heart and Liver.
Sheep heart was dissected into right and left atria, right and left ventricles, and septum. Several random strips from each area were fixed in 4% PFA, cut into 1 x 1-mm cubes, and embedded in OCT medium. 710-µm cryosections were probed with a human-specific antiheat shock protein 27 (HSP27) Ab (Stressgen) (30) or the antidystrophin Ab NCL DYS2 (Novocastra) and antiprotein gene product 9.5 (PGP 9.5, ubiquitin c terminal hydroxylase) (Biogenesis). The secondary Ab for anti-HSP27 and antidystrophin was goat antimouse conjugated to Alexa 488, and the secondary Ab for anti-PGP 9.5 was goat antirabbit conjugated to Alexa 647 (Molecular Probes). Livers of the sheep were fixed in buffered formalin and embedded in paraffin. Liver sections (2 µm) were dewaxed and incubated for 10 min at 90°C for target retrieval. After blocking endogenous peroxidase by EnVision blocking reagent (Dako), sections were incubated in serum-free protein block (Dako) for 10 min and for 2 h in TBS containing 0.1% gelatin and the primary Ab antihuman serum albumin (clone HSA-11; 1:100; Sigma-Aldrich) or monoclonal antihuman hepatocyte Ab (clone OCH1E5; Dako). After washing, sections were incubated for 30 min with labeled polymer (EnVision System; Dako), and immunoreactivity was visualized by incubation with diaminobenzidine tetrahydrochloride. For microdissection applying the PALM Micro Beam System, immunohistochemistry was performed as described above with the monoclonal antihuman hepatocyte, clone OCH1E5 Ab except that sections were mounted on foil-laminated glass slides.
Single Cell PCR Analysis of Fusion/Cell Hybrids.
Isolation of single cells of human origin from human and sheep chimera liver tissue sections with >20% human cells and single cells of ovine origin from chimera liver tissue sections were performed using the PALM Micro Beam System (P.A.L.M. Microlaser Technologies). After target cell identification (the chimeric sheep liver slides were stained previously with the human hepatocyte specific Ab) and dissection from the surrounding tissue by a nitrogen laser beam (LMM), another strong laser was used to catapult (LPC) the microdissected material directly into the tube cap containing 20 µl of PCR buffer (Promega). Before PCR amplifications, cells were digested by proteinase K as described (31). To analyze individual micromanipulated parenchymal liver cells from human liver and human (stained) and ovine (unstained) cells from chimeric sheep liver tissue, genomic fragments of the human VH1, ovine VH7 and human TCRVß7.2 and ovine TCRC
genes were amplified with all external primer pairs in a first PCR round. 1-µl aliquots from the first amplification round were then subjected to a second round of fully nested PCR amplification using internal PCR primers. The sequences of the primers used for PCR amplification are given in Table S2, available at http://www.jem.org/cgi/content/full/jem.20040440/DC1. Single-cell PCR amplifications were performed in a 60-µl reaction mix volume containing 2.5 mM MgCl2 [Promega], 200 µM of each dNTP, 2.5 µM of each primer, 1x PCR buffer [Promega], and 2.5 U platinum Taq-polymerase (Invitrogen). Cycling conditions for the first round included a single 2-min denaturation step at 95°C followed by 34 cycles at 95°C for 1 min, 56°C for 30 s, and 72°C for 1 min, and a 5-min incubation at 72°C. For the second PCR round, 45 cycles of amplification were used. To rule out interspecies cross-reactivity of the primer pairs used, we also amplified DNA from ovine cells with human-specific primers and vice versa. Amplification products were analyzed by agarose gel electrophoresis.
Western Blot for Human Albumin.
Serum proteins (human 50 ng/lane, ovine 500 ng/lane) were separated on a 12% SDS-polyacrylamide gel. After tank blotting onto a nitrocellulose membrane (Amersham Biosciences), blots were blocked with 5% blocking-grade nonfat dry milk (Amersham Biosciences) in PBS containing 0.05% Tween (PBS-T), incubated at 4°C for
12 h with a human albumin-specific Ab (HAS-11, 1:5,000; Sigma-Aldrich). After five washes for 5 min in PBS-T, blots were incubated with horseradish peroxidasecoupled antimouse secondary Ab (1:2,000; Amersham Biosciences) at RT for 1 h. Membranes were washed again and developed using the Amersham Biosciences ECL system according to manufacturer's instructions.
Online Supplemental Material.
Expansion kinetic of USSCs (cell numbers, passages, and population doublings) are shown in Table S1. The sequences of the primers used for PCR amplification in the single cell PCR analysis of fusion/cell hybrids of the chimeric liver tissue are given in Table S2. Tables S1 and S2 are available at http://www.jem.org/cgi/content/full/jem.20040440/DC1.
 |
Results
|
---|
Isolation, Expansion, and Characterization of USSCs from Placental CB.
USSCs were generated from 94 CB samples of a mean gestational age of 39.5 ± 1.46 wk (range 3442), a net volume of 80 ± 21.4 ml including 29 ml citrate-phosphate dextrose (range 43140 ml) and a total nucleated cell count of 7.6 ± 3.1 x 108 (range 2.419.4). No correlation was detected between gestational age (>30 wk), hours elapsed after CB collection, volume, number of mononuclear cells in the CB after gradient separation and the success in generation of USSC. After 625 d (mean 15 d), adherently growing cells (USSC colonies) of fibroblastic morphology were detected with a median frequency of four colonies per CB (range 111). As shown in Fig. 1, A and B, USSCs are adherent, spindle-shaped cells, and have a size of 2025 µm. Hematopoietic CD45+ cells were no longer detected after three passages. The USSC karyotype was normal 46XX or 46XY as analyzed for six individual USSC specimens for passages 5 (21 population doublings) to 19 (45 population doublings). USSCs were negative for CD14, CD33, CD34, CD45, CD49b, CD49c, CD49d, CD49f, CD50, CD62E, CD62L, CD62P, CD106, CD117, glycophorin A, and HLA-DR and expressed high levels of CD13, CD29, CD44, CD49e, CD90, CD105, vimentin, and cytokeratin 8 and 18, human Endo, low levels of CD10, and FLK1 (KDR), and showed variable but weak expression of HLA-ABC (Fig. 1 C). USSCs can be cultured for >20 passages equivalent to >40 population doublings without any spontaneous differentiation (Fig. 1 D and Table S1). The average telomere length of USSCs obtained after both 21 (passage 4) and 25 population doublings (passage 6) was 8.93 kbp; after 36 population doublings, the average telomere length of USSCs obtained was 8.60 kbp (passage 13). This is significantly longer than the telomere length of MSCs generated from a BM donor (age 30 yr) with 7.27 (passage 4) and 7.11 kbp (passage 9) at 19 and 27 population doublings, respectively (Fig. 1 E). USSCs showed expression of transcripts for epidermal growth factor receptor, platelet-derived growth factor receptor, insulin-like growth factor receptor, runt related transcription factor (Runx1), YB1, CD49e, and CD105 (Fig. 1 F). They were negative for the chondrogenic extracellular protein chondroadherin (Fig. 1 F), the bone-specific markers collagenase X, bone sialoprotein, the liver and pancreas-specific markers Cyp1A1 and PDX-1, and neural markers such as NF, synaptophysin, TH, and glial fibrillary acid protein (not depicted). Preliminary cDNA microarray analysis performed for two individual USSCs and one MSC preparation from human BM suggested differential expression of HAS1, which was only detected in MSCs but not in USSCs. This differential expression of HAS1 was confirmed by RT-PCR (n = 55; unpublished data) and immunochemistry.