Drug – bio-affecting and body treating compositions – Whole live micro-organism – cell – or virus containing – Animal or plant cell
Reexamination Certificate
1995-05-16
2003-05-27
Witz, Jean C. (Department: 1651)
Drug, bio-affecting and body treating compositions
Whole live micro-organism, cell, or virus containing
Animal or plant cell
C424S529000, C424S530000, C424S531000, C435S242000
Reexamination Certificate
active
06569427
ABSTRACT:
TABLE OF CONTENTS
1. Introduction . . .
2. Background of the Invention . . .
2.1. Hematopoietic Stem and Progenitor Cells . . .
2.2. Reconstitution of the Hematopoietic System . . .
2.3. Cryopreservation of Cells . . .
2.4. Gene Therapy . . .
3. Summary of the Invention . . .
3.1. Definitions . . .
4. Description of the Figures . . .
5. Detailed Description of the Invention . . .
5.1. Isolation of Fetal or Neonatal Hematopoietic Stem and Progenitor Cells . . .
5.1.1. Collection of Neonatal Blood . . .
5.1.1.1. Volume . . .
5.1.1.2. Preferred Aspects . . .
5.1.1.2.1. Collection Kit . . .
5.1.1.2.2. Vaginal Delivery of the Term Infant . . .
5.1.1.2.3. Other Circumstances of Birth and Delivery . . .
5.1.1.2.3.1. Premature Birth . . .
5.1.1.2.3.2. Multiple Births . . .
5.1.1.2.3.3. Caesarian Delivery . . .
5.1.1.2.3.4. Complicated Delivery
5.1.1.2.3.5. Abnormal Placenta . . .
5.1.1.2.3.6. Collection from the Delivered Placenta . . .
5.1.1.2.3.7. Medical Conditions of the Mother . . .
5.1.1.2.3.8. Unplanned Delivery . . .
5.1.1.2.4. Recordation of Data . . .
5.1.2. Inspection and Testing of Neonatal Blood . . .
5.1.3. Optional Procedures . . .
5.1.3.1. Enrichment for Hematopoietic Stem and Progenitor Cells: Cell Separation Procedures . . .
5.1.3.2. In Vitro Cultures of Hematopoietic Stem and Progenitor Cells . . .
5.2. Cryopreservation . . .
5.3. Recovering Stem and Progenitor Cells from the Frozen State . . .
5.3.1. Thawing . . .
5.3.2. Optional Procedures . . .
5.4. Examination of Cells Recovered for Clinical Therapy . . .
5.4.1. Identity Testing . . .
5.4.2. Assays for Stem and Progenitor Cells . . .
5.5. Hematopoietic Reconstitution . . .
5.6. Therapeutic Uses . . .
5.6.1. Diseases Resulting from a Failure or Dysfunction of Normal Blood Cell Production and Maturation . . .
5.6.2. Hematopoietic Malignancies . . .
5.6.3. Malignant Solid Tumors of Non-Hematopoietic Origin . . .
5.6.4. Autoimmune Disorders . . .
5.6.5. Gene Therapy . . .
5.6.6. Miscellaneous Disorders Involving Immune Mechanisms . . .
5.7 Generation and Use of Hematopoietic Stem and Progenitor Cell Progeny . . .
6. Examples . . .
6.1. Collection of Human Umbilical Cord Blood and Placental Blood . . .
6.2. Hematopoietic Stem and Progenitor Cells in Collected Cord Blood . . .
6.3. Enrichment for Human Hematopoietic Stem and Progenitor Cells: Cell Separation Procedures . . .
6.3.1. Density Separations . . .
6.3.2. Adherence/Non-Adherence Separation.
6.4. Cryopreservation of Cord Blood Stem and Progenitor Cells . . .
6.5. Cell Thawing . . .
6.6. Human Hematopoietic Stem and Progenitor Cell Assays . . .
6.6.1. CFU-GM Assay . . .
6.6.1.1. Preparation of McCoy's 5A Medium . . .
6.6.1.2. Preparation of Human 5637 Urinary Bladder Carcinoma Cell Line Conditioned Medium . . .
6.6.1.3. Preparation of Murine Pokeweed Mitogen Spleen Cell Conditioned Medium . . .
6.6.2. BFU-E-2 and BFU-E-1/CFU-GEMM Assay . . .
6.6.2.1. Preparation of 2.1% Methyl Cellulose . . .
6.6.2.2. Preparation of Hemin . . .
6.6.2.3. Preparation of Iscove's Modified Dulbecco's Medium . . . .
6.6.3. Stem Cell Colony Forming Unit Assay . . .
6.6.4. Assay of the Proliferative Status of Stem and Progenitor Cells . . .
6.7. Recovery After Freeze-Thawing of Human Hematopoietic Progenitor Cells Derived from Cord Blood . . .
6.8. Calculations of the Reconstituting Potential of Cord Blood . . .
6.9. In Vitro Culture Conditions for Hematopoietic Stem and Progenitor Cells . . .
6.10. Mouse Dissection Protocols . . . 6.10.1. Bone Marrow Dissection . . . 6.10.2. Spleen Dissection . . .
6.11. Hematopoietic Reconstitution of Adult Mice with Syngeneic Fetal or Neonatal Stem Cells . . .
6.11.1. Hematopoietic Reconstitution of Lethally-Irradiated Mice with Stem Cells in Blood of the Near-Term Fetus . . .
6.11.2. Hematopoietic Reconstitution of Mice with a Lesser Volume of Near-Term Fetal Blood But Not with Adult Blood . . .
6.11.3. Hematopoietic Reconstitution with Blood of Newborn Mice in Volumes as Low as Ten Microliters . . .
6.11.4. Hematopoietic Reconstitution with Blood of Newborn Mice in Volumes of 10 or 15 Microliters . . .
6.12 Hematopoietic Reconstitution For Treatment of Fanconi's Anemia . . .
6.13. Flowchart: Description of a Service . . .
1. INTRODUCTION
The present invention is directed to hematopoietic stem and progenitor cells of neonatal or fetal blood, that are cryopreserved, and the therapeutic uses of such stem and progenitor cells upon thawing. Such cells can be therapeutically valuable for hematopoietic reconstitution in patients with various diseases and disorders. In a preferred embodiment, neonatal cells that have been cryopreserved and thawed, can be used for autologous (self) hematopoietic reconstitution.
The invention also relates to methods for collection and cryopreservation of the neonatal and fetal stem and progenitor cells of the invention.
2. BACKGROUND OF THE INVENTION
2.1. Hematopoietic Stem and Progenitor Cells
The morphologically recognizable and functionally capable cells circulating in blood include erythrocytes, neutrophilic, eosinophilic, and basophilic granulocytes, B-, T-, nonB-, non T-lymphocytes, and platelets. These mature cells derive from and are replaced, on demand, by morphologically recognizable dividing precursor cells for the respective lineages such as erythroblasts for the erythrocyte series, myeloblasts, promyelocytes and myelocytes for the granulocyte series, and megakaryocytes for the platelets. The precursor cells derive from more primitive cells that can simplistically be divided into two major subgroups: stem cells and progenitor cells (for review, see Broxmeyer, H. E., 1983, “colony Assays of Hematopoietic Progenitor Cells and Correlations to Clinical Situations,” CRC Critical Reviews in Oncology/Hematology 1(3):227-257). The definitions of stem and progenitor cells are operational and depend on functional, rather than on morphological, criteria. Stem cells have extensive self-renewal or self-maintenance capacity (Lajtha, L. G., 1979, Differentiation 14:23), a necessity since absence or depletion of these cells could result in the complete depletion of one or more cell lineages, events that would lead within a short time to disease and death. Some of stem cells differentiate upon need, but some stem cells or their daughter cells produce other stem cells to maintain the precious pool of these cells. Thus, in addition to maintaining their own kind, pluripotential stem cells are capable of differentiation into several sublines of progenitor cells with more limited self-renewal capacity or no self-renewal capacity. These progenitor cells ultimately give rise to the morphologically recognizable precursor cells. The progenitor cells are capable of proliferating and differentiating along one, or more than one, of the myeloid differentiation pathways (Lajtha, L. G. (Rapporteur), 1979, Blood Cells 5:447).
Stem and progenitor cells make up a very small percentage of the nucleated cells in the bone marrow, spleen, and blood. About ten times fewer of these cells are present in the spleen relative to the bone marrow, with even less present in the adult blood. As an example, approximately one in one thousand nucleated bone marrow cells is a progenitor cell; stem cells occur at a lower frequency. These progenitor and stem cells have been detected and assayed for by placing dispersed suspensions of these cells into irradiated mice, and noting those cells that seeded to an organ such as the spleen and which found the environment conducive to proliferation and differentiation. These cells have also been quantified by immobilizing the cells outside of the body in culture plates (in vitro) in a semi-solid support medium such as agar, methylcellulose, or plasma clot in the presence of culture medium and certain defined biomolecules or cell populations which produce and release these molecules. Under the appropriate growth conditions, the stem or progenitor cells will go through a catenated sequence of proliferation and differentiation yielding mature end stage progen
Boyse Edward A.
Broxmeyer Hal E.
Douglas Gordon W.
Pennie & Edmonds LLP
PharmaStem Therapeutics, Inc.
Witz Jean C.
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