WO2007115216A1 - Reprogrammation de cellules souches testiculaires d'humains adultes en cellules souches pluripotentes de la lignee germinale - Google Patents

Reprogrammation de cellules souches testiculaires d'humains adultes en cellules souches pluripotentes de la lignee germinale Download PDF

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WO2007115216A1
WO2007115216A1 PCT/US2007/065709 US2007065709W WO2007115216A1 WO 2007115216 A1 WO2007115216 A1 WO 2007115216A1 US 2007065709 W US2007065709 W US 2007065709W WO 2007115216 A1 WO2007115216 A1 WO 2007115216A1
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cells
cell
stem cells
human
pluripotent
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Chauncey B. Sayre
Francisco J. Silva
Kwok-Yuen F. Pau
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Primegen Biotech Llc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
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    • C12N2500/00—Specific components of cell culture medium
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C12N2501/392—Sexual steroids

Definitions

  • the present invention relates to the field of therapeutically reprogrammed cells.
  • human therapeutically reprogrammed cells are provided that are not compromised by the aging process, are immunocompatible and will function in the appropriate post-natal cellular environment to yield functional cells after transplantation.
  • Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to four divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluhpotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus. At the next stage, cells become multipotent, meaning they can give rise to several other cell types, but those Docket No. 51314-00040
  • hematopoietic cells blood cells that can develop into several types of blood cells, but cannot develop into brain cells.
  • At the end of the long chain of cell divisions that make up the embryo are "terminally differentiated" cells - cells that are considered to be permanently committed to a specific function.
  • stem cells There are three main groups of stem cells; (i) adult or somatic (post-natal), which exist in all post-natal organisms, (ii) embryonic, which can be derived from a pre- embryonic or embryonic developmental stage and (iii) fetal stem cells (pre-natal), which can be isolated from the developing fetus.
  • Each group of stem cells has their own advantages and disadvantages for cellular regeneration therapy, specifically in their differentiation potential and ability to engraft and function de novo in the appropriate or targeted cellular environment.
  • lineage-committed progenitor stem cells and lineage-uncommitted pluripotent stem cells which reside in connective tissues providing the post-natal organism the cells required for continual organ or organ system maintenance and repair. These cells are termed somatic or adult stem cells and can be quiescent or non-quiescent. Typically adult stem cells share two characteristics: (i) they can make identical copies of themselves for long periods of time (long-term self renewal); and (ii) they can give rise to mature cell types that have characteristic morphologies and specialized functions.
  • Somatic stem cells isolated from human bone marrow transferred in utero into pre- immune sheep fetuses have the ability to xenograft into multiple tissues.
  • mesenchymal stem cells which have a wide range of non- hematopoietic differentiation abilities, including bone, cartilage, adipose, tendon, lung, muscle, marrow stroma, and brain tissues.
  • neural stem cells, pancreatic, muscle, adipose, ovarian and spermatogonial stem cells have been found. The therapeutic utility of somatic or post-natal stem cells has been demonstrated and realized through the use of bone marrow transplants.
  • adult somatic stem cells have genomes that have been altered by aging and cell division. Aging results in an accumulation of free radical insults, or oxidative damage, that can predispose the cell to forming neoplasms, reduce cell differentiation ability or induce apoptosis. Repeated cell division is directly related to telomere shortening which is the ultimate cellular clock that determines a cell's functional life-span. Consequently, adult somatic stem cells have genomes that have sufficiently diverged from the physiological prime state found in embryonic and prenatal stem cells.
  • stem cells must be induced to mature into the organ or cell type desired to be useful as therapeutics.
  • the factors affecting stem cell maturation in vivo are poorly understood and even less well understood ex vivo.
  • present maturation technology relies on serendipity and biological processes largely beyond the control of the administering scientist or recipient.
  • embryonic stem (ES) cells as a source of totipotent or pluripotent immunologically privileged cells for use in cellular regenerative therapy.
  • embryonic stem cells themselves may not be appropriate for direct transplant as they form teratomas after transplant, they are proposed as "universal donor" cells that can be differentiated into customized pluripotent, multipotent or committed cells that are appropriate for transplant.
  • moral and ethical issues associated with the isolation of embryonic stem cells from human embryos.
  • GSC germ-line stem cells
  • SSCs spermatogonial stem cells
  • GSCs pluripotent germ-line stem cells
  • the present invention provides biologically useful pluripotent therapeutically reprogrammed cells, generated from adult human stem cells which are suitable for therapeutic applications.
  • Gonadal stem cells may be an ideal stem cell source for cell replacement therapy and therapeutic cloning after reprogramming to pluripotent germ-line stem cells (GSCs).
  • GSCs pluripotent germ-line stem cells
  • SSCs spermatogonial stem cells isolated from the testis can be reprogrammed to form GSC cell lines.
  • GSCs exhibit pluripotent markers, differentiate into embryoid bodies and cells of all three germ layers in vitro, and form chimeric cell populations after incorporation into mouse embryos.
  • GSCs contain long telomerase repeats; they are self-renewing and do not form teratomas after transplantation.
  • a method for therapeutic reprogramming comprising isolating a human adult stem cell; contacting said human adult stem cell with a medium comprising stimulatory factors which induce development of said stem cell into a therapeutically reprogrammed cell; recovering said therapeutically reprogrammed cell from said medium.
  • the human adult stem cell is isolated from the testes.
  • the human adult stem cell is a spermatogonial stem cell.
  • the medium comprises PM-10TM medium.
  • the therapeutically reprogrammed cell is matured into a more terminally differentiated cell.
  • the more terminally differentiated cell is a cardiac myocyte.
  • more terminally differentiated cell is a neural cell.
  • the method further comprises the step of cultuhng the therapeutically reprogrammed cell to form a cell line. Docket No. 51314-00040
  • the method further comprises the step of implanting the therapeutically reprogrammed cell, or a cell matured therefrom, into a host in need of a therapeutically reprogrammed cell.
  • a pluripotent therapeutic composition comprising a therapeutically reprogrammed human adult stem cell.
  • the human adult stem cell is isolated from the testes.
  • the therapeutically reprogrammed human adult stem cell is produced according to the therapeutic reprogramming method.
  • a pluripotent therapeutic composition comprising a therapeutically reprogrammed human adult stem cell which has been induced into a more terminally differentiated cell prior to implantation into a host in need of a therapeutically reprogrammed cells.
  • FIG. 1 depicts the morphologies of adult human testicular isolates in culture on ultra-low adhesive dishes (A), gelatin-coated dishes (B), fibronectin-coated dishes (C) or mouse embryonic feeder cell (MEF)-coated dishes (D) after culture for different periods of time (panels 1 -4) in serum-free PM-10TM medium according to the teachings of the present invention.
  • FIG. 2 depicts the expansion of a regrogrammed adult human testicular stem cell (AHTSC) population in the presence of serum and expression of pluripotent stem cell markers Oct-4 and Nanog according to the teachings of the present invention.
  • FIG. 2A AHTSCs plated on gelatin and cultured for 46 days in serum-free PM-10TM medium contained colonies (arrows and insert) growing on spindle-like cells; the scale bar is 200 ⁇ m;
  • FIG. 2B Rapid cell growth was observed after 2 days of serum addition;
  • FIG. 2C The proliferation rate of AHTSCs is depicted in the presence of serum ( ⁇ ) or serum Docket No. 51314-00040
  • FIG. 3 depicts stem cell markers expressed in a AHTSC population with normal karyotype according to the teachings of the present invention.
  • FIG. 3A Profile of surface markers on AHTSCs that resemble profile on spermatogonial and mesenchymal stem cells, but not on hematopoetic stem cells. Open histograms indicate appropriate isotype control, shaded histograms depict specific antibody staining. Numbers identify the percentage of positive cells.
  • FIG. 4 depicts the expression of pluripotent (Oct-4, Nanog, Dppa ⁇ and Rex- 1 ), gonadal (DAZL) and control ( ⁇ -actin) genes in testicular tissues and isolates by reverse transcriptase-polymerase chain reaction (RT-PCR) before and during culture (between 17 and 42 days) according to the teachings of the present invention.
  • FIG. 5 depicts the expression of pluripotent markers Oct-4 (FIG. 5A), Nanog (FIG. 5B), Oct-4 + Nanog (FIG. 5C), alkaline phosphatase (FIG. 5D), TRA-1 -60 (FIG. 5E) and human mitochondrial protein (FIG. 5F) by spermatogonial stem cells during reprogramming at four weeks after isolation according to the teachings of the present invention.
  • FIG. 6 depicts the spontaneous differentiation of spermatogonial stem cells into cardiomyocytes after more than 30 days in culture and expression of cardiac specific markers troponin-1 (FIG. 6A), cardiac myosin (FIG. 6B), cardiac ⁇ -actin (FIG. 6C) and cardiac ⁇ -actin and human mitochondrial protein (hMP) according to the teachings of the present invention.
  • FIG. 6E depicts the expression of markers by differentiated cells determined by RT-PCR. Docket No. 51314-00040
  • FIG. 7 depicts the culture of testicular cells on fibronectin-coated surfaces for more than 30 days and their spontaneous differentiation into neural cells according to the teachings of the present invention.
  • the cells were assayed for expression of neural- specific markers including MAP-2C (FIG. 7A), NF-160 (FIG. 7B), GFAP and hMP (FIG. 7C-D at two magnifications), Oil Red (FIG. 7E) and nestin (FIG. 7F).
  • FIG. 7G depicts the expression of markers by differentiated cells determine by RT-PCR.
  • FIG. 8 depicts AHTSCs differentiated into mesodermal and endodermal lineages in vitro according to the teachings of the present invention.
  • FIG. 8A Osteogenic induction: Alizarin Red staining of control (left image) and induced AHTSCs (19 days after induction, right image). Scale bar is 400 ⁇ M.
  • FIG. 8B Expression of osteo-specific genes in AHTSCs after induction.
  • FIG. 8C Chondrogenic induction: Alcian Blue staining of control (left image) and induced AHTSCs (13 days after induction, right image). Scale bar is 400 ⁇ M.
  • FIG. 8D Expression of chondro-specific genes in AHTSCs after induction.
  • FIG. 8A Osteogenic induction: Alizarin Red staining of control (left image) and induced AHTSCs (19 days after induction, right image). Scale bar is 400 ⁇ M.
  • FIG. 8B Expression of osteo-specific genes in AHTSCs after induction.
  • FIG. 8C
  • FIG. 8E Adipogenic induction: Oil Red staining of control (left image) and induced AHTSCs (22 days after induction, right image). Scale bar is 25 ⁇ M.
  • FIG. 8F Expression of hepatocyte-specific genes in AHTSCs after induction.
  • FIG. 8G Cardiogenic induction: confocal images of immunofluorescent staining with antibodies specific to cardiocytes after 38 days of induction. Scale bar is 100 ⁇ M.
  • FIG. 8H Expression of cardio-specific genes in AHTSCs after induction.
  • FIG. 9 depicts the differentiation of AHTSCs into the neural lineage cells in vitro according to the teachings of the present invention.
  • FIG. 9A AHTSCs expressed progenitor (nestin), neuronal (Tuj-lll/ ⁇ -tubulin-3, MAP2, NeuN, NFL, NF160) and glial (GFAP, MBP, GaIC) markers after neural induction protocol as assayed by immunocytochemistry. Scale bar for all images is 100 ⁇ M.
  • FIG. 10 depicts the induction of neurogenesis in adult human testicular isolates by growth factors critical for embryonic neuroectoderm formation according to the teachings of the present invention, lmmunohistochemistry was performed on cells Docket No. 51314-00040
  • FIG. 10E depicts the expression of markers by differentiated cells determined by RT-PCR.
  • FIG. 11 depicts transplanted AHTSCs identified by the staining of a human- specific nucleic protein (HuNu) in the spinal cord of uninjured NOD/SCID mice 21 days after transplantation according to the teachings of the present invention.
  • the upper panel shows AHTSCs in the white matter that were double stained with HuNu (labeled with AlexaFluor ® 488) and Tuj-lll (labeled with AlexaFluor ® 568) (FIGs. 11 A-B).
  • the lower panel shows grey matter with transplanted AHTSCs (HuNu) that did not express the astroglial marker GFAP (FIGs. 11 C-D). Scale bars are 50 ⁇ M (FIGs. 11A and 11 C) and 20 ⁇ M (FIGs. 11 B and 11 D).
  • the arrows point to double-stained HuNu+/Tuj-lll+ AHTSCs
  • FIG. 12 depicts AHTSCs in injured spinal cord of NOD/SCID mice 35 days after transplantation according to the teachings of the present invention.
  • Transplanted human cells were identified by immunofluorescence staining with an antibody against human nuclei (HuNu, FIG. 12A-H).
  • Confocal microscopy demonstrates the double labeling of HuNu with oligodendrocyte markers NG2 (FIG. 12A-B) and GaIC (FIG. 12C- D) and neuronal markers Tuj-lll (FIG. 12E-F) and MAP-2 (FIG. 12G-H), all in red.
  • Arrows indicate double-stained AHTSCs. Scale bar is 50 ⁇ M for the left panel and 20 ⁇ M for the right panel.
  • FIG. 13 depicts AHTSCs transplanted into the injured spinal cord of NOD/SCID mice express the neuronal/oligodendroglial progenitor marker A2B5 according to the teachings of the present invention.
  • FIG. 13A Double staining of AHTSCs with HuNu antibody (examples are indicated by arrows) and DNA dye TO- PRO-3. Mouse cells showed DNA stain only.
  • FIG. 13B TO-PRO-3 staining only of the same field as in FIG. 13A showing that AHTSCs have large nuclei (arrows) without bright speckles, a prominent attribute of mouse nuclei. These characteristics allow distinguishing between human and mouse nuclei using DNA-specific stain.
  • FIG. 13C Double staining with A2B5 (labeled with AlexaFluor ® 488) and TO-PRO-3 showing Docket No. 51314-00040
  • FIG. 13D TO-PRO-3 stain only of the same field as in FIG. 13C showing human (examples are indicated by arrows) and mouse nuclei. Scale bar for all images is 20 ⁇ M
  • FIG. 14 depicts AHTSCs transplanted into the injured spinal cord of NOD/SCID mice which express neuronal marker Tuj-lll and increase Tuj-lll expression on host mouse cells at the injury site according to the teachings of the present invention.
  • Control mice received either human foreskin fibroblasts (HFF) or vehicle.
  • FIG. 14A Transplanted AHTSCs identified by anti-Human Mitochondria (HM) antibody (labeled with AlexaFluor ® 488) co-expressed Tuj-lll (labeled with AlexaFluor ® 568).
  • FIG. 14B Transplanted HFF did not show Tuj-lll co-expression.
  • FIG. 14C Mouse cells expressed elevated levels of Tuj-lll around the epicenter of the injury site (indicated by dotted lines) in spinal cord transplanted with AHTSCs in comparison with HFF (FIG. 14D). Scale bars are 20 ⁇ M (FIGs. 14A-B) and 200 ⁇ M (FIGs. 14C-D).
  • FIG. 15 depicts the reduced functional deficits in mice transplanted with AHTSCs into the injured spinal cord of NOD/SCID mice according to the teachings of the present invention.
  • Control mice received either human foreskin fibroblasts (HFF) or vehicle (DMEM).
  • FIG. 15A Basso Mouse Scoring showing significant differences in functional activity between AHTSC-transplanted cohorts and vehicle control cohorts at 28, 35 and 42 days post-injury (DPI). Transplantation started after 6 days of spinal cord injury as indicated by arrow (Day 7).
  • FIG. 15B Kinematic assay on hindlimb stride width in experimental animals (the same groups as in FIG. 15A).
  • FIG. 15C Representative images from hematoxylin and eosin (H&E) stained spinal cords from all three experimental groups showing reduced tissue loss in AHTSCs and HFF transplanted animals.
  • FIG. 15D Morphometric analysis on tissue sections 1 mm either side of the injury epicenter indicates that the spinal cord size in AHTSC cohorts was significantly different from the vehicle control group. * p ⁇ 0.05; ** p ⁇ 0.01
  • Committed refers to cells which are considered to be permanently committed to a specific function. Committed cells are also referred to as “terminally differentiated cells.”
  • Differentiation refers to the adaptation of cells for a particular form or function. In cells, differentiation leads to a more committed cell.
  • Embryonic Stem Cell refers to any cell that is totipotent and derived from a developing embryo that has reached the developmental stage to have attached to the uterine wall. In this context embryonic stem cell and pre-embryonic stem cell are equivalent terms.
  • Embryonic stem cell-like (ESC-like) cells are totipotent or pluhpotent cells not directly isolated from an embryo. ESC-like cells can be derived from primordial sex cells that have been dedifferentiated in accordance with the teachings of the present invention.
  • Fetal Stem Cell refers to a cell that is multipotent and derived from a developing multi-cellular fetus that is no longer in early or mid-stage organogenesis.
  • Germ Cell refers to a reproductive cell such as a spermatocyte or an oocyte, or a cell that will develop into a reproductive cell.
  • Multipotent refers to cells that can give rise to several other cell types, but those cell types are limited in number.
  • An example of a multipotent cell is a hematopoietic cell - a blood stem cell that can develop into several types of blood cells but cannot develop into brain cells.
  • Multipotent adult progenitor cells refers to multipotent cells isolated from the bone marrow which have Docket No. 51314-00040
  • Pluripotent refers to cells that can give rise to any cell type except the cells of the placenta or other supporting cells of the uterus.
  • Pluripotent Germ Stem Cell As used herein "pluripotent germ stem cell” or “PGS” refers to a primordial sex cell that has been therapeutically reprogrammed to be pluripotent and can be maintained in culture.
  • Post-natal Stem Cell refers to any cell that is multipotent and derived from a multi-cellular organism after birth.
  • Primordial Sex Cell refers to any diploid cell that is derived from the male or female mature or developing gonad, is able to generate cells that propagate a species and contains a diploid genomic state. Primordial sex cells can be quiescent or actively dividing. These cells include male gonocytes, female gonocytes, spermatogonial stem cells, ovarian stem cells, oogonia, type-A spermatogonia, Type-B spermatogonia. Primordial sex cells are also known as germ-line stem cells (GSC).
  • GSC germ-line stem cells
  • Primordial germ cell refers to cells present in early embryogenesis that are destined to become germ cells.
  • Reprogramming refers to the resetting of the genetic program of a cell such that the cell exhibits pluripotency and has the potential to produce a fully developed organism.
  • Somatic Stem Cells As used herein, “somatic stem cells” refers to diploid multipotent or pluripotent stem cells. Somatic stem cells are not totipotent stem cells.
  • Therapeutic Reprogramming refers to the process of maturation wherein a stem cell is exposed to stimulatory factors according to the teachings of the present invention to yield either pluripotent, multipotent or tissue-specific committed cells. Therapeutically reprogrammed cells are useful for implantation into a host to replace or repair diseased, damaged, defective or genetically Docket No. 51314-00040
  • the therapeutically reprogrammed cells of the present invention do not possess non-human sialic acid residues.
  • Totipotent refers to cells that contain all the genetic information needed to create all the cells of the body plus the placenta. Human cells have the capacity to be totipotent only during the first few divisions of a fertilized egg.
  • the present invention provides biologically useful pluripotent therapeutically reprogrammed cells, generated from adult human stem cells are suitable for therapeutic applications.
  • Therapeutic reprogramming refers to a maturation process wherein a stem cell is exposed to stimulatory factors according the teachings of the present invention to yield pluripotent, multipotent or tissue-specific committed cells.
  • the process of therapeutic reprogramming can be performed with a variety of stem cells including, but not limited to, therapeutically cloned cells, hybrid stem cells, embryonic stem cells, fetal stem cells, multipotent post-natal stem cells (adult progenitor cells), adipose-derived stem cells (ADSC) and primordial sex cells.
  • Stem cells are primitive cells that give rise to other types of cells. Also called progenitor cells, there are several kinds of stem cells. Totipotent cells are considered the "master" cells of the body because they contain all the genetic information needed to create all the cells of the body plus the placenta, which nourishes the human embryo. Human cells have this totipotent capacity only during the first few divisions of a fertilized egg. After three to four divisions of totipotent cells, there follows a series of stages in which the cells become increasingly specialized. The next stage of division results in pluripotent cells, which are highly versatile and can give rise to any cell type except the cells of the placenta or other supporting tissues of the uterus.
  • cells become multipotent, meaning they can give rise to several other cell types, but those types are limited in number.
  • An example of a multipotent cell is a hematopoietic cell - a Docket No. 51314-00040
  • primordial germ cells Upon migration and colonization of the genital ridge, the primordial germ cells undergo differentiation into male or female germ cell precursors (primordial sex cells).
  • primordial sex cells For the purpose of this invention disclosure, only male primordial sex cells (PSC) will be discussed, but the qualities and properties of male and female primordial sex cells are equivalent and no limitations are implied.
  • primordial stem cells become closely associated with precursor Sertoli cells leading to the beginning of the formation of the seminiferous cords.
  • primordial germ cells When the primordial germ cells are enclosed in the seminiferous cords, they differentiate into gonocytes that are mitotically quiescent. These gonocytes divide for a few days followed by arrest at G 0 /Gi phase of the cell cycle. In mice and rats these gonocytes resume division within a few days after birth to generate spermatogonial stem cells and eventually undergo differentiation and meiosis related to spermatogenesis.
  • Primordial sex cells are directly responsible for generating the cells required for fertilization and eventually a new round of embryogenesis to create a new organism. Primordial sex cells are not programmed to die and are of a quality comparable to that of an embryonic state. Docket No. 51314-00040
  • Embryonic stem cells are cells derived from the inner cell mass of the pre- implantation blastocyst-stage embryo and have the greatest differentiation potential, being capable of giving rise to cells found in all three germ layers of the embryo proper. From a practical standpoint, embryonic stem cells are an artifact of cell culture since, in their natural environment in the epiblast, they only exist transiently during embryogenesis. Manipulation of embryonic stem cells in vitro has lead to the generation and differentiation of a wide range of cell types, including cardiomyocytes, hematopoietic cells, endothelial cells, nerves, skeletal muscle, chondrocytes, adipocytes, liver and pancreatic islets. Growing embryonic stem cells in co-culture with mature cells can influence and initiate the differentiation of the embryonic stem cells to a particular lineage.
  • an embryo and a fetus are distinguished based on the developmental stage in relation to organogenesis.
  • the pre-embryonic stage refers to a period in which the pre-embryo is undergoing the initial stages of cleavage.
  • Early embryogenesis is marked by implantation and gastrulation, wherein the three germ layers are defined and established.
  • Late embryogenesis is defined by the differentiation of the germ layer derivatives into formation of respective organs and organ systems.
  • the transition of embryo to fetus is defined by the development of most major organs and organ systems, followed by rapid fetal growth.
  • Fetal stem cells have been isolated from the fetal bone marrow (hematopoietic stem cells), fetal brain (neural stem cells) and amniotic fluid (pluripotent amniotic stem cells). In addition, stem cells have been described in both adult male and fetal tissues. Fetal stem cells serve multiple roles during the process of organogenesis and fetal development, and ultimately become part of the somatic stem cell reserve.
  • Maturation is a process of coordinated steps either forward or backward in the differentiation pathway and can refer to both differentiation and/or dedifferentiation.
  • a cell, or group of cells interacts with its cellular environment during embryogenesis and organogenesis. As maturation progresses, cells begin to form niches and these niches, or microenvironments, house stem cells that direct and regulate organogenesis. At the time of birth, maturation has Docket No. 51314-00040
  • a single stem cell clone can contribute to generations of lineages such as lymphoid and myeloid cells for more than a year and therefore have the potential to spread mutations if the stem cell is damaged.
  • the body responds to a compromised stem cell by inducing apoptosis thereby removing it from the pool and preventing potentially dysfunctional or tumorigenic properties.
  • Apoptosis removes compromised cells from the population, but it also decreases the number of stem cells that are available for the future. Therefore, as an organism ages, the number of stem cells decrease. In addition to the loss of the stem cell pool, there is evidence that aging decreases the efficiency of the homing mechanism of stem cells.
  • Telomeres are the physical ends of chromosomes that contain highly conserved, tandem-repeated DNA sequences. Telomeres are involved in the replication and stability of linear DNA molecules and serve as counting mechanism in cells; with each round of cell division the length of the telomeres shortens and at a pre-determined threshold, a signal is activated to initiate cellular senescence. Stem cells and somatic cells produce telomerase, which inhibits shortening of telomeres, but their telomeres still progressively shorten during aging and cellular stress. Docket No. 51314-00040
  • stem cells can be differentiated into particular cell types in vitro and shown to have the potential to be multipotent by engrafting into various tissues and transit across germ layers and as such have been the subject of much research for cellular therapy.
  • immune rejection is the limiting factor for cellular therapy.
  • the recipient individual's phenotype and the phenotype of the donor will determine if a cell or organ transplant will be tolerated or rejected by the immune system.
  • the present invention provides methods and compositions for providing functional immunocompatible stem cells for cellular regenerative/reparative therapy.
  • the therapeutic reprogramming method of the present invention is suitable for reprogramming cells from a variety of animals including, but not limited to, primates, rodents, sheep, cattle, goats, pigs, horses, etc.
  • the primate is a human.
  • Therapeutic reprogramming takes advantage of the fact that certain stem cells are relatively easily to obtain, such as spermatogonial stem cells and adipose- derived stem cells, and epigenetically reprograms these cells by exposure to stimulatory factors. These therapeutically reprogrammed cells have changed their maturation state to either a more committed cell lineage or a less committed cell lineage. Therapeutically reprogrammed cells are therefore capable of repairing or regenerating disease, damaged, defective or genetically impaired tissues.
  • Therapeutic reprogramming uses stimulatory factors, including without limitation, chemicals, biochemicals and cellular extracts to change the epigenetic Docket No. 51314-00040
  • primordial sex cells are therapeutically reprogrammed.
  • Primordial sex cells residing in the lining of the seminiferous tubules of the testes and the lining of the ovaries (the spermatogonia and oogonia, respectively) have been determined to possess diploid (2N) genomes remarkably undamaged by to the effects of aging and cell division.
  • 2N diploid
  • PSCs possess genomes in a nearly physiologically prime state.
  • a non-limiting example of a PSC particularly useful in an embodiment of the present invention is a spermatogonial stem cell.
  • therapeutically reprogrammed PSC cells are prepared for the maturation process using means similar to that experienced by stem cells present in the developing embryo and fetus during embryogenesis and organogenesis.
  • spermatogonial stem cells adult human testicular stem cells
  • human testicular stem cells isolated from human males undergoing standard castration surgical procedures and who have been on long-term hormone (estrogen) treatment
  • spermatogonial stem cells are therapeutically reprogrammed by culture in the presence of cell growth promoting and maintenance and maturation factors.
  • a series of culture media have been developed by the present inventors which contain cell growth promoting, maintenance, reprogramming and maturation factors for the therapeutic reprogramming of post-natal stem cells. These media are disclosed in co-pending United States Patent Application No. 11/488,362 which is incorporated by reference herein for all it contains regarding media.
  • the reprogramming medium is PM- 10TM.
  • PM-10TM media contains the signals necessary for human spermatogonial stem cells to be therapeutically reprogrammed into pluripotent embryonic stem cell-like cells.
  • the cell growth and maturation factors useful for the therapeutic reprogramming of PSCs using PM-10TM media in a serum-free environment include, but are not limited to, epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2), glial cell derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF).
  • EGF epidermal growth factor
  • FGF2 fibroblast growth factor 2
  • GDNF glial cell derived neurotrophic factor
  • LIF leukemia inhibitory factor
  • the therapeutically reprogrammed human cells made in accordance with the teachings of the present invention are useful in a wide range of therapeutic applications for cellular regenerative/reparative therapy.
  • the therapeutically reprogrammed human cells of the present invention can be used to replenish stem cells in mammals whose natural stem cells have been depleted due to age or ablation therapy such as cancer radiotherapy and chemotherapy.
  • the therapeutically reprogrammed human cells of the present invention are useful in organ regeneration and tissue repair.
  • therapeutically reprogrammed human cells can be used to reinvigorate damaged muscle tissue including dystrophic muscles and muscles damaged by ischemic events such as myocardial infarcts.
  • the therapeutically reprogrammed human cells disclosed herein can be used to ameliorate scarring in animals, including humans, following a traumatic injury or surgery.
  • the therapeutically reprogrammed human cells of the present invention are administered systemically, such as intravenously, and migrate to the site of the freshly traumatized tissue recruited by circulating cytokines secreted by the damaged cells.
  • the therapeutically reprogrammed human cells can be administered locally to a treatment site in need or repair or regeneration.
  • murine spermatogonial stem cells can be reprogrammed to germ-line stem cells (GSCs) that express GFP (a marker of Oct-4 expression) and pluhpotent markers, including Oct-4, Nanog, SSEA-1 and alkaline phosphatase.
  • GFP a marker of Oct-4 expression
  • pluhpotent markers including Oct-4, Nanog, SSEA-1 and alkaline phosphatase.
  • the GFP + murine GSC cell lines have been propagated without losing the expression of pluripotent markers and telomerase activity.
  • these murine GSCs form embryoid bodies, differentiate into neural, adipose and cardiac phenotypes, incorporate into the inner cell mass of recipient mouse embryos, and form chimeric cell populations in the heart, lung, liver and brain.
  • the present inventors disclose herein the derivation of GSCs from adult human testicular isolates which express pluripotent markers during reprogramming, differentiate into multi-lineages, and form multipotent cell lines.
  • One requirement of stem cell therapy is a constant cell supply. While ES and EG cells multiply indefinitely, adult stem cells have a finite renewal limitation.
  • the present inventors have established human pluripotent cell lines from the gonad that are self-renewal and transplantable.
  • the germ-line stem cells of the present application are multipotent, expressing several pluripotent stem cell markers and exhibiting differentiation potential to form lineages of all three germ layers. Furthermore, upon transplantation of neural cell differentiated from adult human testicular stem cells (GSCs) into the site of a spinal cord injury in NOD/SCID mice, the AHTSCs survived, integrated into the host tissue, expressed neuronal and oligodendoglial markers and reduced functional deficits. The transplanted cells do not demonstrate tumorogenic activity.
  • GSCs adult human testicular stem cells
  • a cell culture medium for therapeutically reprogramming human stem cells comprising a cell culture growth medium base; a plurality of vitamins and minerals and a plurality of cell growth and maturation factors.
  • the cell culture medium is serum free.
  • the therapeutic reprogramming cell culture medium comprises a plurality of cell growth and maturation factors selected from the group consisting of recombinant human epidermal growth factor, recombinant human fibroblast growth factor 2, recombinant human glial cell derived neurotrophic factor and human leukemia inhibitory factor.
  • the recombinant human epidermal growth factor is present at a concentration of between approximately 10 ng/mL and approximately 40 ng/mL, preferably approximately 20 ng/mL.
  • the recombinant human fibroblast growth factor 2 is present at a concentration of between approximately 1 ng/mL and approximately 120 ng/mL.
  • the recombinant human glial cell derived neurotrophic factor is present at a concentration of between approximately 2 ng/mL and approximately 40 ng/mL, preferably between approximately 10 ng/mL and approximately 20 ng/mL.
  • the human leukemia inhibitory factor is present at a concentration of between approximately 1 ,000 units/mL and approximately 10,000 units/mL, preferably approximately 1 ,000 units/mL.
  • the cell culture medium is PM-10TM (Table 1 ).
  • rhGDNF neurotrophic factor 1 ,000 units/mL human leukemia 1000 U/mL inhibitory factor (LIF)
  • Testicular samples were washed 5 times in cold phosphate-buffered saline (PBS) containing 0.01 % EDTA. Seminiferous tubules were dissected, minced, and digested with collagenase/DNase. Dissociated cells were centrifuged and re- suspended in the serum-free PM-10TM medium containing a stem cell medium base, non-essential amino acids, and 19 cell growth-promoting and reprogramming factors, including GDNF. Blood and somatic cells were discarded after overnight differential adhesion. Stem cells in suspension were collected and plated onto culture dishes coated with gelatin, fibronectin, mouse feeder cells, or low-adhesive surface. In average, about 150 million cells per testis were obtained. The plating density was adjusted to approximately 10 6 cells per 7 cm 2 . Cells and cell aggregates were culture on the PM-10TM medium for 40 days during which time multiple samples were collected for pluripotent marker assays.
  • PBS cold phosphate-buffered sa
  • AHTSCs were maintained in serum-free PM-10TM medium for several months when plated onto either gelatin or fibronectin substrates (FIG. 2A).
  • the morphology of these cultures consisted of colonies (arrows and insert in FIG. 2A) growing on clusters of spindle-like cells apparently as outgrowths of the colonies.
  • Addition of serum or serum replacement to the culture promoted cell division within days (FIG. 2B).
  • the estimated rate of doubling cell numbers was every 26 hours and the AHTSC population was propagated every 10 days (FIG. 2C).
  • the AHTSC population stained positive for the pluripotent markers, Oct-4 (FIG. 2D) and Nanog (FIG. 2E).
  • the propagated cells maintained their marker characteristics for at least 6 passages (see Fig. 3 below). No change of morphology or growth patterns was observed after freezing and thawing for up to 12 passages.
  • Oct-4 is a signature of pluripotent cells; its expression disappears as pluripotent cells commit to a cell lineage and differentiate. In a commercially available adult human testicular RNA sample, no Oct-4 expression was Docket No. 51314-00040
  • FIG. 4, lane 2 Similarly in adult human testis samples before therapeutic reprogramming (FIG. 4, lanes 4-7), no Oct-4 expression was detected. In contrast, Oct- 4 expression was up-regulated by 17-42 days of culture in suspension or on adhesive surfaces (FIG. 4, lanes 8-11 ).
  • a non-human primate ESC sample (FIG. 4, lane 3; known to express Oct-4 and Rex-1 ) was included as positive control.
  • Several markers found in pluripotent ESCs, including Nanog, Dppa ⁇ and Rex-1 were expressed in adult human testicular tissues and isolates (FIG. 4). Morphologically, cells in cultures grown on fibronectin (FIG.
  • FIG. 5A-F and gelatin exhibited pluripotent protein markers by immunocytochemical staining of Oct-4 and Nanog (FIG. 5A-C), alkaline phosphatase (FIG. 5D) and TRA-1 -60 (FIG. 5E).
  • FIG. 5F The identity of human cells on MEF co-cultures were confirmed by staining the human-specific mitochondria protein (FIG. 5F).
  • suspended cells, cell aggregates and/or attached colonies were isolated either manually or by a brief collagenase treatment (1 mg/mL for 15 min). They were then plated onto mouse embryonic feeder cells or fibronectin coverslips surrounded by mouse embryonic feeder cells.
  • FIG. 6D As well as Nkx2.5, GATA-4, cardiac ⁇ -actin and myosin, but not atrial natriuretic peptide (ANP) (FIG. 6E).
  • Nkx2.5 a cardiomyocyte-specific marker
  • FIG. 7A-F cardiomyocytes
  • fibronectin-coated coverslips showed morphologies different from cardiomyocytes (FIG. 7A-F). These cells attached within 2 weeks and subsequently differentiated into phenotypes that expressed neural lineage markers vimentin, nestin and NeuroDI , as well as neuronal and glial markers, including NF-68/160, MAP-2C, GAD67, GFAP and MBP (FIG. 7G).
  • the expression of MAP-2C (FIG. 7A), NF160 (FIG. 7B), GFAP+hMP (FIG. 7C and 7D, two magnifications) and nestin (FIG. 7F) were also shown by immunohistochemistry.
  • GFAP which is expressed only in cells plated onto fibronectin-coated coverslips (FIG. 7G)
  • the expression of neural progenitor and phenotype markers was found in cells plated onto fibronectin-coated coverslips in dishes covered without or with human feeders or MEFs (the human origin was confirmed by the expression of hMP).
  • the AHTSC population can be induced by neural protocols containing growth factors platelet derived growth factor (PDGF), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF), sonic hedgehog (SHH), fibroblast growth factor 8 (FGF-8) and brain derived neurotrophic factor (BDNF) to differentiate into multiple neural phenotypes.
  • PDGF platelet derived growth factor
  • FGF-2 fibroblast growth factor 2
  • EGF epidermal growth factor
  • SHH sonic hedgehog
  • FGF-8 fibroblast growth factor 8
  • BDNF brain derived neurotrophic factor
  • cells were plated onto gelatin-coated (0.2%) plastic dishes or coverslips in DMEM low glucose supplemented with Glutamax, penicillin/streptomycin in the presence or absence of different concentrations of FBS and growth factors according to the specific differentiation protocol as described below.
  • AHTSCs were plated onto fibronectin-coated coverslips and incubated in DMEM/F-12 with the addition of N2 supplements (Invitrogen), PDGF 10 ng/ml, FGF-2 10 ng/ml, and EGF 20 ng/ml for 4-14 days.
  • N2 supplements Invitrogen
  • PDGF 10 ng/ml fibronectin-coated coverslips
  • FGF-2 10 ng/ml fibronectin-coated coverslips
  • EGF 20 ng/ml for 4-14 days.
  • AHTSCs For induction of AHTSCs into osteocytes, cells were plated onto gelatin- coated dishes and cultured in DMEM low glucose with Glutamax, penicillin/streptomycin, 20% FBS, dexamethazone 10OnM, ascorbic acid 0.25mM and ⁇ - glycerolphosphate 1 OmM for up to 25 days.
  • AHTSCs were cultured in the Chondrogenic SingleQuotsTM media (Cambrex) with the addition of 20% FBS and TGF-3 ⁇ (IOng/ml,). The induction time was 14 days. The efficiency of differentiation was determined by Alcian Blue stain and RT-PCR for chondrogenic marker expression.
  • adipocyte differentiation cells were plated onto gelatin-coated dishes and cultured during several repeating cycles consisting of induction media for the first 3 days following by maintenance media for 1 day.
  • the induction medium was DMEM with low glucose, Glutamax, penicillin/streptomycin, 20% FBS, dexamethasone 1 ⁇ M, isobutylmethylxanthine 0.5 mM, indomethacin 200 ⁇ M and insulin 10 ⁇ M.
  • the Maintenance medium was DMEM with low glucose, Glutamax, penicillin/streptomycin, 20% FBS and insulin 10 ⁇ M. After 20-25 days of induction, successful adipogenesis was confirmed by Oil Red staining.
  • hepatocyte differentiation cells were plated onto Mathgel ® -coated plates.
  • the induction medium consisted of DMEM low glucose with MCDB-201 40% (Sigma), Glutamax, penicillin/streptomycin, 5%FBS, and was supplemented with ITS+LA-BSA (10 ⁇ g/ml insulin, 5.5 ⁇ g/ml human transferrin, 5 ng/ml sodium selenite, 0.5 mg/ml bovine serum albumin, 4.7 ⁇ g/ml linoleic acid, dexamethasone 1 nM, ascorbic acid 100 ⁇ M, FGF-4 10 ng/ml, and HGF 20 ng/ml.
  • Hepatocyte specific marker expression was identified by PCR
  • AHTSCs expressed markers indicative of neural progenitor cells (nestin, vimentin), neuronal cells (NeuN, NF-L, and NF160, tuj-lll/ ⁇ -tubulin3 and MAP2), glial cells (GFAP, MBP, GaIC), but not maturing Schwann cells (S-100) (FIG. 9A-B). Moreover, some cells expressed the dopaminergic marker, Nurr-1.
  • AHTSCs can also be induced to differentiate into phenotypes of the mesodermal and endodermal lineages expressing markers that are indicative of cardiomyocytes, chondrocytes, osteocytes, adipocytes, or hepatocytes (FIG. 8).
  • SHH sonic hedgehog
  • FGF-8 fibroblast growth factor 8
  • TGF- ⁇ transforming growth factor ⁇
  • PGDF-BB platelet derived growth factor BB
  • the supplement of growth factors includes sonic hedgehog (150 ng/ml), fibroblast growth factor (FGF)-8 (75 ng/ml), platelet-derived growth factor (PDGF)-BB (20 ng/ml), and transforming growth factor (TGF)- ⁇ 3 (4 ng/ml); some are involved in neural progenitor cell formation in the isthmus (or the mid-hindbrain boundary).
  • the cells were cultured in this medium for 40 days with medium changes every other day.
  • the effect of SHH and/or FGF-8 may be critical for glial cell maturation since cells cultured in the control medium with TGF- ⁇ and PDGF-BB but without SHH and FGF-8 did not express the glial markers GFAP and MBP (FIG. 10E).
  • pluripotent cells such as ES cells or embryonal germ (EG) cells, were known to be capable of differentiating into all three germ layers.
  • Crush injuries were performed under Avertin anesthesia (0.6 ml/20 g).
  • Age matched female NOD/SCID mice (Taconic) were used for all animal studies.
  • a midline incision of the skin was made over T6-L2 and the paravertebral muscles were separated from the T8-T10 vertebrae.
  • T9 dorsal laminectomy the column was stabilized, and a T9 crush injury was performed by stabilizing the vertebral column, grasping the spinal cord with a fine forceps (FST) calibrated to 0.4mm and holding the forceps closed for 5 seconds.
  • FST fine forceps
  • Animals were placed on soft bedding over a heating pad held at 37°C for 3 hours and given nourishment. Animals were behaviorally assessed 1 and 6 days following spinal cord injury and randomized to receive cellular transplantations.
  • animals were videotaped using a Canon Digital Video Camcorder ZR500 from underneath plexiglass bearing defined 1cm grid lines. The videos were analyzed frame by frame using Microsoft Windows Movie Maker software. Analysis consisted of rear paw stride length, defined as the distance from the start of a step with the rear paw thru to the end of that step with the same paw (measurements taken on each side for three Docket No. 51314-00040
  • stride width defined as the distance from the left outermost hind paw digit to the right outermost hind paw digit as previously described 40 .
  • All behavioral analysis was conducted one day prior to injury, then on days 1 , 6, 8, 14, 21 , 28, 35, and 42 following spinal cord injury. All behavioral analysis was done by scorers blinded to the different transplantation groups.
  • One and 6 days following SCI animals were BMS assessed and randomized to receive cellular transplantation.
  • Tuj-lll FIG. 11A and 11 B
  • GFAP astroglial marker
  • AHTSCs, human foreskin fibroblasts (HFF), and vehicle control (DMEM) were transplanted into NOD/SCID mice following spinal cord injury and the cords were evaluated 35 days post transplantation. Mice were anesthetized and transcardially perfused with 60 ml of 4% paraformaldehyde. Spinal cords were dissected out of the vertebral column and postfixed overnight in 4% paraformaldehyde. Cords were then placed into 25% sucrose/PBS until sunk. A block of cord from T4 to L3 was embedded longitudinally in OCT compound and frozen at -2O 0 C for sectioning. 10 ⁇ m thick longitudinal sections were serially placed onto slides as to each section on one slide was 200 ⁇ m apart from the following on that same slide.
  • MicrosuiteTM-Five basic edition analysis software (Olympus), using all tissue sections in which the central canal was visible. The tissue diameters at each 100 ⁇ m interval for each animal within a group were averaged.
  • Transplanted human cells were identified by staining of human nuclei (HuNu; FIG. 12). Many AHTSCs were found in or at the vicinity of the transplantation site (FIG. 12A, 12C, 12E and 12G, low magnification). The AHTSC expressing oligodendroglial or neuronal markers was identified by double staining of NG2/HuNu, GalC/HuNu, Tuj- lll/HuNu or MAP-2/HuNu (indicated by arrows in FIG. 12B, 12D, 12F and 12H, in higher magnification).
  • the average cross-sectional area 1 mm rostral and caudal to the injury epicenter was 0.586 ⁇ 0.19mm 2 for the neural-enriched AHTSC transplanted cohort, 0.441 ⁇ 0.11 mm 2 for the HFF transplanted cohort, and 0.21 O ⁇ O.02mm 2 for the vehicle control cohort.

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Abstract

La présente invention concerne des procédés destinés à programmer à des fins thérapeutiques des cellules souches d'adulte humain pour en faire des cellules pluripotentes. Des cellules programmées à des fins thérapeutiques à partir de cellules testiculaires adultes sont décrites. Les cellules reprogrammées à des fins thérapeutiques peuvent être utilisées dans une thérapie de régénération cellulaire et elles ont le pouvoir de se différencier en lignées de cellules plus engagées.
PCT/US2007/065709 2006-03-30 2007-03-30 Reprogrammation de cellules souches testiculaires d'humains adultes en cellules souches pluripotentes de la lignee germinale WO2007115216A1 (fr)

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US11/488,362 US20070020759A1 (en) 2004-07-15 2006-07-17 Therapeutic reprogramming of germ line stem cells
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EP2090649A1 (fr) 2008-02-13 2009-08-19 Fondazione Telethon Procédé de reprogrammation de cellules différentiées
EP2176399A1 (fr) * 2007-08-07 2010-04-21 PrimeGen Biotech LLC Isolement, caractérisation et propagation de cellules souches germinales
US9657267B2 (en) 2009-11-05 2017-05-23 Primegen Biotech Llc Ex host maturation of germline stem cells

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EP2176399A1 (fr) * 2007-08-07 2010-04-21 PrimeGen Biotech LLC Isolement, caractérisation et propagation de cellules souches germinales
EP2090649A1 (fr) 2008-02-13 2009-08-19 Fondazione Telethon Procédé de reprogrammation de cellules différentiées
US9657267B2 (en) 2009-11-05 2017-05-23 Primegen Biotech Llc Ex host maturation of germline stem cells

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