WO2006071794A2 - Postpartum cells derived from umbilical cord tissue, and methods of making and using the same - Google Patents

Postpartum cells derived from umbilical cord tissue, and methods of making and using the same Download PDF

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WO2006071794A2
WO2006071794A2 PCT/US2005/046851 US2005046851W WO2006071794A2 WO 2006071794 A2 WO2006071794 A2 WO 2006071794A2 US 2005046851 W US2005046851 W US 2005046851W WO 2006071794 A2 WO2006071794 A2 WO 2006071794A2
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cell
cells
protein
alpha
derived
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WO2006071794A3 (en
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Ian Ross Harris
Darin J. Messina
Anthony Kihm
Agneiszka Seyda
David Colter
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Ethicon Inc
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Priority to EP05855417.1A priority patent/EP1831356B1/en
Priority to AU2005322060A priority patent/AU2005322060B2/en
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    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
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Definitions

  • the cells can grow under a variety of environmental conditions.
  • the cells can grow under a wide range of atmospheric conditions.
  • Presently preferred are atmospheres which range from about 5% O 2 to about 20% or more O 2 .
  • the cells grow and expand well in Growth Medium under these conditions, typically in the presence of about 5% CO 2 , and the balance of the atmosphere as nitrogen.
  • the skilled artisan will appreciate that the cells may tolerate broader ranges of conditions in different media, and that optimization for specific purposes may be appropriate.
  • the cells can be shown to produce several or more of the CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha, and HLA-A,B 5 C and concomitantly not produce one, or several, or more of CD31, CD34, CD45, CDl 17, CD141, or HLA-DR 5 DP 3 DQ as detected by flow cytometry. More highly preferred are cells that produce each of CDlO, CD 13, CD44, CD73 5 CD90, PDGFr-alpha, and HLA-A,B,C and concomitantly not produce any of CD31, CD34, CD45, CDl 17, CD141, or HLA-DR 5 DP 5 DQ as detected by flow cytometry.
  • the isolation procedure also utilizes an enzymatic digestion process.
  • Many enzymes are known in the art to be useful for the isolation of individual cells from complex tissue matrices to facilitate growth in culture.
  • a broad rE ⁇ ge df ⁇ digestive" enzyrri'es-' ⁇ f use'in cell isolation from tissue is available to the skilled artisan. Ranging from weakly digestive (e.g. deoxyribonucleases and the neutral protease, dispase) to strongly digestive (e.g. papain and trypsin), such enzymes are available commercially.
  • antithrombogenic agents such as antithrombogenic agents, anti-apoptotic agents, and anti-inflammatory agents may be useful and may be administered in sequence with, or coadministered with the cells, individually or in combinations or two or more such compounds or agents.
  • anti-apoptotic agents may be useful to minimize programmed cell death.
  • agents include but are not limited to EPO, EPO derivatives and analogs, and their salts, TPO, IGF-I, IGF-II, hepatocyte growth factor (HGF), and caspase inhibitors.
  • Kits for assays and in vitro methods as described herein may contain one or more of (1) UDCs or fractions, components or products of UDCs, (2) reagents for practicing the in vitro method, (3) other cells or cell populations, as appropriate, for example for cocultures and (4) instructions for conducting the in vitro method.
  • the markers for PDGFreceptor alpha and HLA-ABC are '-felfereia/TMs ⁇ Mstibtty 6FtHeT cellfe with respect to their cell surface markers allows the cells to be engineered in certain respects to provided full advantage of the cells desirous properties and to minimize any unwanted or deleterious properties relating to cell surface markers. This is particularly useful in engineering or preparing cells for implantation or for use in grafting where adverse immunological reactions and rejection are concerns.
  • therapeutic cultures comprising postpartum-derived cells expanded in serum- free medium
  • Such cells have any useful properties and are more readily suited for commercial application and regulatory approval having, for example, little risk of disease transmission related to foreign animal serum or proteins.
  • the therapeutic cultures comprise cells having a signature gene profile wherein mRNA from genes for reticulon, oxidized LDL receptor, and IL-8 cells are grown in medium containing serum or medium free of serum.
  • Such cultures may comprise a single cell type or may consist of multiple cell types including PPDCs and other cell types as additional cells present in the co- or mixed culture.
  • the cells isolated from umbilical cord tissues were seeded at 5,000 cells/cm 2 onto gelatin-coated T-75 flasks (Corning Inc., Coming, NY) in Growth Medium. After two days, spent medium and unadhered cells were aspirated from the flasks. Adherent cells were washed with PBS three times to remove debris and blood-derived cells. Cells were then replenished with Growth Medium and allowed to grow to confluence (about 10 days from passage 0) to passage 1. On subsequent passages (from passage 1 to 2 etc), cells reached sub-confluence (75-85 percent confluence) in 4-5 days. For these subsequent passages, cells were seeded at 5,000 cells/cm . Cells were grown in a humidified incubator with 5 percent carbon dioxide at 37 0 C.
  • the lysate was centrifuged (10 minutes at 200 x g).
  • the cell pellet was resuspended in Complete Minimal Essential Medium (Gibco, Carlsbad CA) containing 10 percent fetal bovine serum (Hyclone, Logan UT), 4 millimolar glutamine (Mediatech Herndon, VA ), penicillin at 100 Units per milliliter and streptomycin at 100 micrograms per milliliter (Gibco, Carlsbad, CA).
  • the resuspended cells were centrifuged (10 minutes at 200 x g), the supernatant was aspirated, and the cell pellet was washed in complete medium.
  • Umbilicus-derived cells were isolated as described in a previous example. Cells were seeded at 1,000 cells/cm and passaged as described above until senescence. Cells were grown under standard atmospheric conditions at 37 0 C. Growth Medium was changed twice per week. Cells were passaged as they reached about 85% confluence. At each passage, cells were trypsinized and counted by Trypan blue staining.
  • Table 2-1 Growth characteristics for different cell populations grown to senescence
  • the medium was replaced with a modified Growth Medium (DMEM with D- valine (special order Gibco), 15% (v/v) dialyzed fetal bovine serum (Hyclone, Logan, UT), 0.001% (v/v) betamercaptoethanol (Sigma), penicillin at 50 Units/milliliter and streptomycin at 50 milligrams/milliliter (Gibco)).
  • DMEM modified Growth Medium
  • D- valine special order Gibco
  • 15% v/v dialyzed fetal bovine serum
  • betamercaptoethanol Sigma
  • penicillin 50 Units/milliliter
  • streptomycin at 50 milligrams/milliliter
  • Umbilicus-derived postpartum cells are positive for CDlO, CD13, CD44, CD73, CD90, PDGFr-alpha, HLA-A 5 B 5 C and negative for CD31, CD34, CD45, CDl 17, CD141 and HLA-DR 5 DP 5 DQ. This identity was consistent between variations in variables including the donor, passage, culture vessel surface coating, and digestion enzymes used in isolation and preparation of the cells.
  • cytokine receptor CRL2 precursor [Homo sapiens] transforming growth factor, beta 2 hypothetical protein MGC29643 antigen identified by monoclonal antibody MRC OX-2 putative X-linked retinopathy protein
  • Gene expression profiles of cells derived from the human umbilical cord were compared with those of cells derived from other sources using an Affymetrix GENECHIP.
  • Six "signature” genes were identified: oxidized LDL receptor 1, interleukin-8 (IL-8), renin, reticulon, chemokine receptor ligand 3 (CXC ligand 3), and granulocyte chemotactic protein 2 (GCP-2). These "signature” genes were expressed at relatively high levels in umbilicus-derived cells.
  • Genes identified by cDNA microarray as uniquely regulated in postpartum cells were further investigated using real-time and conventional PCR.
  • Immunohistochemistry was performed similar to previous studies (e.g., Messina, et al. (2003) Exper. Neurol. 184: 816-829). Tissue sections were washed with phosphate-buffered saline (PBS) and exposed to a protein blocking solution containing PBS, 4% (v/v) goat serum (Chemicon, Temecula, CA), and 0.3% (v/v) Triton (Triton X-100; Sigma) for 1 hour to access intracellular antigens. In instances where the epitope of interest would be located on the cell surface (CD34, ox-LDL Rl), triton was omitted in all steps of the procedure in order to prevent epitope loss.
  • PBS phosphate-buffered saline
  • Triton Triton X-100
  • Vimentin, desmin, alpha-smooth muscle actin, cytokeratin 18, von Willebrand Factor, and CD34 are produced in cells within human umbilical cord.
  • Stimulator (donor) allogeneic PBMC, autologous PBMC, and postpartum cell lines were treated with mitomycin C.
  • Autologous and mitomycin C-treated stimulator cells were added to responder (recipient) PBMCs and cultured for 4 days. After incubation, [ 3 H]thymidine was added to each sample and cultured for 18 hours. Following harvest of the cells, radiolabeled DNA was extracted, and [ 3 H]-thymidine incorporation was measured using a scintillation counter. Reactions were performed in triplicate using two-cell culture plates with three receivers per plate
  • Cells derived from the postpartum umbilical cord are useful for regenerative therapies.
  • Tissue produced by SCID mice following transplantation of a biodegradable material with and without umbilicus-derived cells was evaluated.
  • the materials evaluated were VNW nonwoven scaffolds, 35/65 PCL/PGA foam, and a self-assembling peptide hydrogel.
  • mice Male mice ⁇ Mas musculus) (Fox Chase SCID; Harlan Sprague Dawley, Inc., Indianapolis, Indiana), were used at 5 weeks of age. All handling of the SCID mice took place under a hood. Each animal was individually weighed, and anesthetized with an intraperitoneal injection of a mixture of 60 milligram/kilogram KETASET (ketamine hydrochloride) (Aveco Co., Inc., Fort Dodge, Iowa), 10 milligram/kilogram ROMPUN (xylazine) (Mobay Corp., Shawnee, Kansas) and saline.
  • KETASET ketamine hydrochloride
  • ROMPUN xylazine
  • Table 14-3 Staining results for human nestin, GFAP, and TuJl respectively in Two Stage Differentiation Experiment. Note that + means that at least a portion (> 0%) of the cells were positive for the stain indicated.
  • Human nestin immature neural stem and progenitor cells
  • GFAP astrocytes
  • TuJl immature and mature neurons.
  • Angiogenesis or the formation of new vasculature, is necessary for the growth of new tissue. Induction of angiogenesis is an important therapeutic goal in many pathological conditions.
  • the angiogenic activity of umbilicus-derived cells in in vitro assays was examined. A well-established method of assessing angiogenic activity involving seeding endothelial cells onto a culture plate coated with a basement membrane extract (Nicosia and Ottinetti (1990) In Vitro CellDev. Biol. 26(2): 119-28), was utilized. Treating endothelial cells on such basement membranes or extracellular matrix material with angiogenic factors will stimulate the cells to form a network that is similar to capillaries.
  • Table 17-1 shows quantities of known angiogenic factors released by UDCs in Growth Medium at atmospheric oxygen conditions.
  • UDCs were seeded onto inserts as described above. The cells were cultured at 37°C in atmospheric oxygen for 48 hours on the inserts and then switched to a 2% FBS media and returned at 37°C for 24 hours. Medium was removed, immediately frozen, and stored at -80 0 C, and analyzed by the Searchlight multiplex ELISA assay (Pierce Chemical Company, Rockford, IL). Results shown are the averages of duplicate measurements.
  • HNF-I alpha a hepatocyte-specific transcription factor
  • cytoplasmic intermediate filament proteins such as keratin 19 (Kl 9), keratin 8 (K8), and cytokeratin 18 (CKl 8)
  • Kl 9 keratin 19
  • K8 keratin 8
  • CKl 8 cytokeratin 18
  • albumin and cytochrome p450 2B6 were selected as markers for hepatocyte differentiation (Schwartz et al. (2002) J. CHn. Invest. 109(10):1291-1302; Okumoto et al. (2003) Biochem. Biophys. Res. Commun. 304(4):691-695;
  • Umbilicus-derived cells P4
  • Umbilicus-derived cells P9
  • neonatal and adult NHDF Cells were seeded at a density of 5,000 cells/cm 2 in T25 flasks in Chang C medium (Irvine Scientific, Santa Ana, CA) on either fibronectin (PeproTech, Rocky Hill, NJ) or gelatin (Sigma) and grown for two passages until confluence. Cells were then seeded at 1,000 cells/cm 2 in 24- well TCP plates and grown as described above until they reached about 40-60% confluence.
  • Oil Red O Staining Cells were fixed with 10 percent (v/v) neutral buffered formalin (Richard-Allan Kalamazoo, MI). After fixation, the cells were washed in deionized water and incubated for two minutes in propylene glycol (absolute; Poly Scientific, Bay Shore, NY). Propylene glycol was removed by aspiration, and samples were incubated in Oil Red O (Poly Scientific) for one hour. Staining solution was removed by aspiration and stained samples were then incubated in 85 percent (v/v) propylene glycol solution (Poly Scientific) for one minute. Stained samples were washed with two changes of de-ionized water.
  • Beta II medium Equal parts of DMEM/Ham's F12 media, 2% FBS, 10 millimolar nicotinamide, 25 millimolar glucose; and
  • EBM Endothelial Cell Basal Medium
  • Human mesenchymal stem cells (Lot# 2Fl 656) were obtained from Biowhittaker, Walkersville, MD and were cultured in MSCGM according to manufacturers instructions. This lot has been tested previously, and was shown to be positive in the chondrogenesis assays.
  • Human adult and neonatal fibroblasts were obtained from American Type Culture Collection (ATCC), Manassas, VA and cultured in Growth Medium on gelatin-coated tissue culture plastic flasks. Postpartum tissue-derived cells, isolated from human umbilical cords as described in previous examples, were utilized. Cells were cultured in Growth Medium in a manner similar to the culture of the fibroblasts. The cell cultures were incubated at 37°C with 5% CO 2 . Cells used for experiments were at passages 3 and 12.
  • Fractional shortening values were calculated as described by Sahn et al. (1978) Circulation 58:1072-1083.
  • the fractional shortening of the vehicle-treated animals had a significant decrease from 47.7% ⁇ 8.3% at Day 0 to 23.5% ⁇ 30.2% at Day 28 (p ⁇ 0.05).
  • the animals that were treated with umbilicus-derived cells showed small, non-significant differences between the fractional shortening between Day 0 and 28. There were no significant differences between the fractional shortening between the treatment groups at Day 0.
  • V max from fitting the data with a Naka-Rushton curve was not used because ERG responses were often erratic at higher luminance levels in dystrophic animals and showed tendencies for depressed responses around 0.4 and 1.4 log candila/m 2 .
  • criterion amplitudes were used: 20 micro Volts for a- and b-waves, and 10 micro Volts for STR-like responses. The amplitude of the b-wave was measured from the a-wave negative peak up to the b-wave positive apex, and not up to the peak of oscillations, which can exceed the b-wave apex (4).
  • Umbilicus-derived cell-transplanted animals demonstrated good improvement in all outcome measures tested at 60 days (Table 25-1), a-wave (27 ⁇ 11) versus sham controls (0), mixed b-wave (117 ⁇ 67) versus sham controls (18 ⁇ 13), cone-b-wave (55 ⁇ 25) versus sham controls (28 ⁇ 11), and in rod contribution (49 ⁇ 16%) versus sham controls (6 ⁇ 7%).

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US8815587B2 (en) 2014-08-26
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