WO2002046373A9 - Method of collecting placental stem cells - Google Patents
Method of collecting placental stem cellsInfo
- Publication number
- WO2002046373A9 WO2002046373A9 PCT/US2001/046506 US0146506W WO0246373A9 WO 2002046373 A9 WO2002046373 A9 WO 2002046373A9 US 0146506 W US0146506 W US 0146506W WO 0246373 A9 WO0246373 A9 WO 0246373A9
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- placenta
- exogenous
- embryonic
- stem cells
- Prior art date
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- C12N5/0602—Vertebrate cells
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- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0605—Cells from extra-embryonic tissues, e.g. placenta, amnion, yolk sac, Wharton's jelly
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions
- Human stem cells are totipotential or pluripotential precursor cells capable of generating a variety of mature human cell lineages. This ability serves as the basis for the cellular differentiation and specialization necessary for organ and tissue development. Recent success at transplanting such stem cells have provided new clinical tools to reconstitute and/or supplement the bone marrow after myeloablation due to disease, exposure to toxic chemical or radiation. Further evidence exists which demonstrates that stem cells can be employed to repopulate many, if not all, tissues and restore physiologic and anatomic functionality. The application of stem cells in tissue engineering, gene therapy delivery and cell therapeutics is also advancing rapidly.
- a method of extracting embryonic-like stem cells from a drained placenta by means of a perfusion technique which utilizes either or both of the umbilical artery and umbilical vein has been developed, on the recovery of human placenta following exsanguination and collection of the residual blood.
- the placenta is then processed in such a manner as to establish an ex vivo, natural bioreactor environment in which resident stem cells within the parenchyma and extravascular space are recruited and migrate into the empty microcirculation where they can be washed into a collecting vessel by perfusion.
- the extraction procedure is based on the passage of the perfusion liquid through either or both of the umbilical artery and umbilical vein using a gravity flow into the placenta which is suspended in such a manner that the umbilical artery and umbilical vein are at the highest point. It is preferred to connect the umbilical artery and the umbilical vein simultaneously as shown in Figure 1 to a pipette that is connected via a flexible connector to a reservoir of the perfusion liquid which is passed into the umbilical vein and artery and collected in a suitable open vessel from the surface of the placenta that was attached to the uterus of the mother during gestation.
- the cells in the placenta can be stimulated to produce bioactive molecules, such as immunoglobulin, or other molecules, or stimulated to proliferate, for example, by administration of erythropoietin.
- the cells can also be genetically engineered prior to harvest, while still in the bioreactor, or at the time of harvest, using for example a viral vector such as an adenoviral or retroviral vector, or using mechanical means such as liposomal or chemical mediated uptake of the DNA.
- Growth factors are usually proteins and include but are not limited to cytokines, lymphokines, interferons, colony stimulating factors (CSF's), interferons, chemokines, and interleukins.
- Other growth factors include recombinant human hemopoietic growth factors including ligands, stem cell factors, thrombopoeitin (Tpo), interleukins, and granulocyte colony-stimulating factor (G-CSF).
- the growth factors introduced into the perfusion solution can stimulate the propagation of undifferentiated stems cells or differentiated hemopoietic cells and stimulate the production of bioactive molecules including but not limited to immunoglobulins, hormones, or other growth factors as previously described.
- phosphate buffered saline solution PBS
- a 10 ml portion is collected and centrifuged for 25 minutes at 3000 rpm (revolutions per minute).
- the effluent is divided into four tubes and placed in an ice bath.
- 2.5 ml of a 1% fetal calf serum (FCS) solution in PBS is added and the tubes are centrifuged (140 minutes x 10 g (acceleration due to gravity)).
- FCS 1% fetal calf serum
- the pellet is resuspended in 5 ml of 1% FCS and two tubes are combined.
- the total mononucleocytes are calculated by adding the total lymphocytes and the total monocytes and multiplying this by the total cell suspension volume.
- Example 2 Analysis of Cells obtained by Perfusion and Incubation of Placenta.
- Total cell number of PP after Ficoll is 5.3X10 s and number of CB before processing is 6.3 X 10 8 .
- Lym% is percent of lymphocytes; MID% is percent of midrange white blood cells; and GRA% is percent of granulocytes.
- Cell isolation is achieved by using magnetic cell separation, such as for example, Auto Macs (Miltenyi).
- CD 34 + cell isolation is performed first.
- Placenta donors were recruited from expectant mothers that enrolled in private umbilical cord blood banking programs and provided informed consent permitting the use of the exsanguinated placenta following recovery of cord blood for research purposes. These donors also permitted use of blinded data generated from the normal processing of their umbilical cord blood specimens for cryopreservation. This allowed comparison between the composition of the collected cord blood and the effluent perfusate recovered using this experimental method described below. All donor data is confidential.
- placenta Following exsanguination of the umbilical cord and placenta, the placenta was placed in a sterile, insulated container at room temperature and delivered to the laboratory within 4 hours of birth. Placentas were discarded if, on inspection, they had evidence of physical damage such as fragmentation of the organ or avulsion of umbilical vessels. Placentas were maintained at room temperature (23+/-2°C) or refrigerated (4°C) in sterile containers for 2 to 20 hours. Periodically, the placentas were immersed and washed in sterile saline at 25+/- 3°C to remove any visible surface blood or debris.
- the umbilical cod was transected approximately 5 cm from its insertion into the placenta and the umbilical vessels were cannulated with Teflon or polypropylene catheters connected to a sterile fluid path allowing bi-directional perfusion of the placenta and recovery of the effluent fluid.
- the system employed in the present invention enabled all aspects of conditioning, perfusion and effluent collection to be performed under controlled ambient atmospheric conditions as well as real-time monitoring of mtravascular pressure and flow rates, core and perfusate temperatures and recovered effluent volumes.
- a range of conditioning protocols were evaluated over a 24 hour post-partum period and the cellular composition of the effluent fluid was analyzed by flow cytometry, light microscopy and colony forming unit assays.
- the placenta was maintained under varying conditions in an attempt to simulate and sustain a physiologically compatible environment for the proliferation and recruitment of residual cells.
- the cannula was flushed with IMDM serum-free medium (GibcoBRL, NY) containing 2U/ml heparin (EJkins-Sinn, NJ).
- Perfusion of the placenta continued at a rate of 50 mL per minute until approximately 150 mL of perfusate was collected. This volume of perfusate was labeled "early fraction”.
- This volume of perfusate was labeled "early fraction”.
- Continued perfusion of the placenta at the same rate resulted in the collection of a second fraction of approximately 150 mL and was labeled "late fraction".
- the placenta was gently massaged to aid in the perfusion process and assist in the recovery of cellular material. Effluent fluid was collected from the perfusion circuit by both gravity drainage and aspiration through the arterial cannula.
- Placentas were obtained from delivery rooms along with cord blood after obtaining written parental consent, and were processed at room temperature within 12 to 24 hours after delivery. Before processing, the membranes were removed and the maternal site washed clean of residual blood. The umbilical vessels were cannulated with catheters made from 20 gauge Butterfly needles use for blood sample collection. Placentas were then perfused with heparinized (2U/mL) Dulbecco's modified Eagle Medium (H.DMEM) at the rate of 15 mL/minute for 10 minutes and the perfusates were collected from the maternal sites within one hour and the nucleated cells counted. The perfusion and collection procedures were repeated once or twice until the number of recovered nucleated cells fell below 100/microL.
- H.DMEM Dulbecco's modified Eagle Medium
- the perfusates were pooled and subjected to light centrifugation to remove platelets, debris and de-nucleated cell membranes.
- the nucleated cells were then isolated by Ficoll-Hypaque density gradient contrifugation and after washing, resuspended in H.DMEM.
- For isolation of the adherent cells aliquots of 5- 10x10 6 cells were placed in each of several T-75 flasks and cultured with commercially available Mesenchymal Stem Cell Growth Medium (MSCGM) obtained from BioWhittaker, and placed in a tissue culture incubator (37° C, 5% CO 2 ). After 10 to 15 days, the non-adherent cells were removed by washing with PBS, which was then replaced by MSCGM.
- the flasks were examined daily for the presence of various adherent cell types and in particular, for identification and expansion of clusters of fibroblastoid cells.
- Cells are recovered from the perfusates by centrifugation at X 00 x g for 15 minutes at room temperature. This procedure served to separate cells from contaminating debris and platelets. The cell pellets were resuspended in IMDM serum-free medium containing 2U/ml heparin and 2mM EDTA (GibcoBRL, NY). The total mononuclear cell fraction was isolated using Lymphoprep (Nycomed Pharma, Oslo, Norway) according to the manufacturer's recommended procedure and the mononuclear cell fraction was resuspended. Cells were counted using a hemocytometer. Viability was evaluated by trypan blue exclusion.
- Isolation of mesenchymal cells was achieved by "differential trypsinization," using a solution of 0.05% trypsin with 0.2% EDTA (Sigma). Differential trypsinization was possible because fibroblastoid cells detached from plastic surfaces within about five minutes whereas the other adherent populations required more than 20-30 minutes incubation. The detached fibroblastoid cells were harvested following trypsinization and trypsin neutralization, using Trypsin Neutralyzing Solution (TNS, BioWhitaker). The cells were washed in H.DMEM and resuspended in MSCGM.
- Flow cytometry was carried out using a Becton-Dickinson FACSCalibur instrument and FITC and PE labeled monoclonal antibodies, selected on the basis of known markers for bone marrow-derived MSC (mesenchymal stem cells), were purchased from B.D. and Caltag laboratories (S.San Francisco, CA.), and SH2, SH3 and Sh4 antibody producing hybridomas were obtained from AM. Cul. and reactivities of the MoAbs in their cultured supernatants were detected by FITC or PE labeled F(ab)'2 goat anti-mouse antibodies. Lineage differentiation was carried out using the commercially available induction and maintenance culture media (BioWhittaker), used as per manufacturer's instructions.
- the placental-derived cells in contrast to their earlier phase, adhered within hours, assumed characteristic fibroblastoid shape, and formed a growth pattern identical to the reference bone marrow-derived MSC.
- the loosely bound mononuclear cells were washed out and the cultures remained homogeneous and devoid of any visible non-fibroblastoid cell contaminants.
- CD-34, CD-38, and other stem cell-associated surface markers on early and late fraction purified mononuclear cells was assessed by flow cytometry. Briefly, cells were washed in PBS and then double-stained with anti- CD34 phycoerythrin and anti-CD38 fluorescein isothiocyanate (Becton Dickinson, Mountain View, CA).
Abstract
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Priority Applications (14)
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KR1020037007561A KR100915482B1 (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells |
CA002430989A CA2430989A1 (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells |
MXPA03005014A MXPA03005014A (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells. |
EP01999637.0A EP1349918B1 (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells |
DK01999637.0T DK1349918T3 (en) | 2000-12-06 | 2001-12-05 | Method of collecting stem cells from placenta |
ES01999637.0T ES2522890T3 (en) | 2000-12-06 | 2001-12-05 | Method to collect placental stem cells |
JP2002548091A JP4294316B2 (en) | 2000-12-06 | 2001-12-05 | Method for collecting placental stem cells |
AU2020902A AU2020902A (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells background of the invention |
AU2002220209A AU2002220209B2 (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells |
IL15630301A IL156303A0 (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells |
EP10183378.8A EP2305795B1 (en) | 2000-12-06 | 2001-12-05 | Method of collecting placental stem cells |
IL156303A IL156303A (en) | 2000-12-06 | 2003-06-04 | Method of collecting placental stem cells from a post-partum human placenta |
IL208510A IL208510A (en) | 2000-12-06 | 2010-10-06 | Isolated nucleated placental cell population |
IL213109A IL213109A (en) | 2000-12-06 | 2011-05-24 | Method of propagating exogenous cells in a placental bioreactor |
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JP (1) | JP4294316B2 (en) |
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