NZ617582B2 - Compositions and methods for autologous germline mitochondrial energy transfer - Google Patents

Abstract

Disclosed is a method of preparing an oocyte for in vitro fertilisation (IVF) or artificial insemination, the method comprising transferring a composition comprising i) oogonial stem cell (OSC) mitochondria, or ii) mitochondria isolated from a progeny of an OSC, the OSC having been isolated from ovarian tissue, into an autologous oocyte, thereby preparing the oocyte for IVF or artificial insemination, wherein the OSC is an isolated non-embryonic stem cell that is mitotically competent and expresses Vasa, Oct-4, Dazl, Stella and optionally a stage-specific embryonic antigen. rian tissue, into an autologous oocyte, thereby preparing the oocyte for IVF or artificial insemination, wherein the OSC is an isolated non-embryonic stem cell that is mitotically competent and expresses Vasa, Oct-4, Dazl, Stella and optionally a stage-specific embryonic antigen.

Description

COMPLETE SPECIFICATION APPLICANT THE GENERAL HOSPITAL CORPORATION TITLE COMPOSITIONS AND METHODS FOR GOUS GERMLINE MITOCHONDRIAL ENERGY TRANSFER COMPOSITIONS AND METHODS FOR AUTOLOGOUS GERMLINE MITOCHONDRIAL ENERGY TRANSFER RELATED APPLICATIONS This ation claims the benefit ofUS. provisional application Ser. No. 61/475,561 filed April 14, 2011 and US. provisional application Ser. No. 61/600,505, filed February 17, 2012, the entire disclosures of which are orated herein by reference.

STATEMENT MADE UNDER LLY SPONSORED RESEARCH This work was supported in part by National utes on Aging Grant No. NIH R37- AG012279 and National Institutes on Health National Research Service Award (F32-AG034809).

BACKGROUND OF THE INVENTION During the past few decades, because of cultural and social changes, women in the developed world have cantly delayed childbirth. For example, first birth rates for women —44 years of age in the United States have increased by more than 8-fold over the past 40 years (Ventura Vital Health Stat 47: 1—27, 1989 Matthews NCHS Data 009 21 :1—8). It is well known that pregnancy rates in women at 35 or more years of age are significantly lower, both naturally and with assisted reproduction. The decline in live birth rate reflects a decline in response to ovarian stimulation, reduced embryo quality and pregnancy rates, and an increased incidence of miscarriages and fetal aneuploidy. In addition, associated chromosomal and meiotic spindle abnormalities in eggs are considered the major factors responsible for the increased incidence of infertility, fetal loss (miscarriage) and conceptions resulting in birth defects — most notably trisomy 21 or Down syndrome — in women at advanced reproductive ages fl-Ienderson et al., Nature 1968 218:22—28, Hassold et al., Hum Genet 1985 17, Battaglia et al., Hum Reprod 1996 11:2217—2222, Hunt et al., Trends Genet 2008 24:86—93).

At present there is no known intervention to improve the pregnancy outcome of older female patients. In animal studies, chronic administration of pharmacologic doses of anti-oxidants during the juvenile period and throughout adult reproductive life has been reported to improve oocyte quality in aging female mice (Tarin et al., Mal Reprod Dev 2002 61:385—397). However, this approach has significant long-term ve effects on ovarian and e fimction, leading to higher fetal death and resorptions as well as decreased litter ncy and size in d animals (Tarin et al., Heriogenology 2002 57:1539—1550). Thus, clinical translation of chronic anti- oxidant therapy for maintaining or improving oocyte quality in aging females is impractical.

Aging and age-related pathologies are frequently associated with loss ofmitochondrial n. due to decreased mitochondrial numbers (biogenesis), diminished mitochondrial activity (production ofATP, which is the main source of energy for cells) and/or lation ofmitochondrial DNA (mtDNA) mutations and deletions. As oocytes age and oocyte mitochondrial energy production decreases, many ofthe critical processes ofoocyte maturation, required to produce a competent egg, especially nuclear spindle ty and chromosomal segregation, become impaired ann et al., JAssist Reprod Genet 2004 21:79—83, Wilding et al., Zygote 2005 13:317-23). logous transfer of cytoplasmic extracts fi'om young donor ooeytes (viz. obtained from different women) into the oocytes ofolder women with a y oductive failure, procedure known as ooplasmic transplantation or mic transfer, demonstrated improved embryo development and delivery oflive offspring. Unforttmately. however, the children born following this procedure exhibit mitochondrial heteroplasmy or the presmce ofmitochondria ‘ from two different sources (Cohen et a1., M01Hum Reprod 1998 4:269—80, Ban-itt et al., Hum Reprod 2001 16:513-6, ton-Hanis et al., Nature 1982 299:460—2, Harvey et al., Gim- Top Dev Biol 2007 77:229-49. This is consistent with the fact that maternally-derived mitochondria present in the egg are used to “seed" the embryo with mitochondria, as paternally- derived mitochondria item the sperm are destroyed shortly after fertilization (Sutovsky et al., BiolReprod 2000 635820590). Although the procedure involves transfer ofcytoplasm and not purified or isolated mitochondria from the donor eggs, the ce ofdonor mitochondria in transferred cytoplasm, confirmed by the passage of“foreign” mitochondria into the o ’ is believed to be the reason why heterologous ooplasmic er provides a fertility benefit.

Irrespective, the health impact ofinduced ondrial heteroplnsmy in these children is as yet tmlmown; r, it has been demonstrated that a mouse model chondrial heteroplasmy produces a phenotype consistent with metabolic syndrome (Acton et al., Biol Reprod 2007 77: 569—76). Arguably, the most significant issue with heterologous OOpIasmic transfer is tied to the fact that mitochondria also contain genetic al that is ct from nuclear genes contributed by the biological mother and biological father.

Accordingly, the children ved following this ure have three genetic parents (biological mother, biological father, egg donor), and thus represent an example of genetic manipulation ofthe human germline for the generation ofembryos. Ooplasmic transplantation procedures that result in mitochondrial heteroplasmy are therefore now regulated and largely prohibited by the FDA. For details, see CBER 2002 Meeting Documents, ical Response Modifiers Advisory Committee minutes from May 9, 2002, which are publicly available from the FDA and “Letter to Sponsors I Researchers - Human Cells Used in Therapy Involving the Transfer of Genetic Material By Means Other Than the Union ofGamete Nuclei", which is also ly ble from the FDA http://www.fda.gov/BiologicsBloodVaccines/SafetyAvailability/ucml05852.htm.

Although the use of autologous mitochondria from somatic cells would avoid mitochondrial heteroplasmy, the mitochondria of somatic cells also suffer from age-related loss of mitochondrial function, due to decreased mitochondrial s (biogenesis), diminished mitochondrial activity (production ofATP, which is the main source of energy for cells) and/or accumulation of mitochondrial mtDNA mutations and deletions. Therefore, for women of advanced maternal age, no significant benefit would have been expected from transferring mitochondria derived from autologous somatic cells into oocytes. Moreover, a variety of stem cells are known to possess low mitochondrial activity (Ramalho—Santos et a1., Hum Reprod Update. 2009 (5):553-72) and, therefore, adult stem cells were not thought to be viable sources of high ty mitochondria.

SUlVIMARY OF THE INVENTION The present invention is based, in part, upon the surprising discovery that the mammalian female germline stem cells or oogonial stem cells (OSCs), which are present in the somatic tissue of the ovary, contain mitochondria with the highest known ATP- generating capacity of all stem cell types evaluated, and containing mtDNA having a d amount of accumulated mutations, including, in some cases, non-detectable levels ofa common mtDNA deletion known to accumulate with age in somatic cells.

In one aspect, the invention provides a method of ing an oocyte for in vitro fertilization (IVF) or artificial insemination. The method comprises transferring a composition comprising OSC mitochondria, or mitochondria ed from a progeny of an OSC, into an autologous oocyte, thereby preparing the oocyte for in vitro fertilization or artificial insemination.

In some embodiments, the OSC is an isolated non-embryonic stem cell that is mitotically competent and expresses Vasa, Oct-4, Dazl, Stella and optionally a stage- specific embryonic antigen (SSEA) (e.g., , -2, -3, and -4). The OSC can be obtained from ovarian tissue, or non-ovarian tissue/sources, such as, e.g., bone marrow or blood, e.g., peripheral and umbilical cord blood.

In other embodiments, the composition comprising OSC mitochondria, or mitochondria ed from a progeny of an OSC, is the cytoplasm of the cells without a In yet other embodiments, the composition sing OSC mitochondria or mitochondria obtained from the y of an OSC is a purified preparation. In certain embodiments, the purified preparation does not contain or is at least about 85%, 90%, 95% free of OSCs, OSC progeny and/or non-functional mitochondria.

In some embodiments, the composition comprises 1x103 to 5x104 mitochondria.

In other embodiments, the OSC or progeny of an OSC produces at least 5—fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or hymal stem cell is autologous.

In yet other embodiments, the OSC or progeny of an OSC produces at least 10- fold more ATP per fg mtDNA than an ovarian c cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In still other embodiments, the OSC or progeny ofan OSC produces at least 50- fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain, ments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In other embodiments, the OSC or progeny of an OSC produces at least IOO—fold more ATP per fg mtDNA than an ovarian somatic cell or hymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some embodiments, the oocyte is obtained from a human female of advanced maternal age. In other embodiments, the oocyte is obtained from a human female with low n reserve.

In some embodiments, the composition ses mitochondria that have been isolated by centrifugation. In other ments, the composition comprises mitochondria that have been isolated by mitochondrial membrane potential-dependent cell sorting.

In some embodiments, the composition comprising mitochondria obtained from at least one OSC or at least one progeny of an OSC, is the cytoplasm of the cells without a nucleus. In other embodiments, the ition comprising mitochondria ed from at least one OSC or at least one progeny of an OSC is a purified preparation of mitochondria.

In a d embodiment there is provided a composition comprising isolated OSC mitochondria, or mitochondria ed from a progeny of an OSC.

In some embodiments, the composition is at least about 85%, 90%, 95% free of cells or non-functional mitochondria.

In some embodiments, the composition comprises 1x103 to 5x104 mitochondria.

In other embodiments, the OSC or progeny of an OSC produces at least 5-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some ments, the OSC or progeny of an OSC produces at least 10- fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In other embodiments, the OSC or progeny of an OSC es at least 50- fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In n embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some embodiments, the OSC or progeny of an OSC produces at least 100-fold more ATP per fg mtDNA than an ovarian c cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some embodiments, the OSC or progeny of an OSC is obtained fiom a human female of advanced maternal age. In other embodiments, the OSC or progeny of an OSC is obtained from a human female with low ovarian reserve.

In some embodiments, the composition comprises ondria that have been IO isolated by fugation. In other embodiments, the composition comprises mitochondria that have been ed by mitochondrial membrane potential-dependent cell sorting.

In yet another , the invention provides a composition comprising at least one isolated mitochondrion obtained from an OSC or at least one progeny of an OSC.

In some embodiments, the composition comprises 1x103 to 5x104 ondria.

In other embodiments, the OSC or progeny ofan OSC es at least 5-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or hymal stem cell is autologous.

In some ments, the OSC or progeny of an OSC produces at least 10-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian c cell or hymal stem cell is autologous.

In other ments, the OSC or progeny of an OSC produces at least 50-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some embodiments, the OSC or progeny of an OSC produces at least 100-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some embodiments, the OSC or progeny of an OSC is obtained from a human female of advanced maternal age. In other embodiments, the OSC or progeny of an OSC is obtained from a human female with low ovarian reserve.

In some embodiments, the composition comprises mitochondria that have been isolated by centrifugation. In other embodiments, the composition comprises mitochondria that have been isolated by mitochondrial membrane potential—dependent cell sorting.

In another aspect there is provided an unfertilized oocyte prepared by a method embodied by the invention.

In another related embodiment there is provided an oocyte comprising exogenous, autologous OSC mitochondria or mitochondria obtained from a progeny of an OSC.

In another related ment there is provided a method of in vitro fertilization.

The method ses the steps of: a) obtaining a composition comprising i) mitochondria obtained from an OSC, or ii) mitochondria obtained from a progeny of an OSC; b) transferring the ition into an isolated, autologous oocyte; and c) fertilizing the gous oocyte in vitro to form a zygote. In an embodiment, the method further comprises transferring the zygote, or a lantation stage embryo derived from the zygote, into the uterus or oviduct of a female subject.

In some embodiments, the composition comprises 1x103 to 5x104 mitochondria.

In other ments, the OSC or progeny of an OSC produces at least 5-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian c cell or mesenchymal stem cell is autologous.

In yet other embodiments, the OSC or progeny of an OSC produces at least 10-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian c cell or mesenchymal stem cell is autologous.

In still other ments, the OSC or progeny of an OSC produces at least 50- fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain, embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In other ments, the OSC or progeny of an OSC produces at least lOO-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some embodiments, the oocyte is obtained from a human female of advanced maternal age. In other embodiments, the oocyte is obtained from a human female with low ovarian reserve.

In some embodiments, the composition comprises mitochondria that have been isolated by centrifugation. In other embodiments, the composition comprises ondria that have been ed by mitochondrial membrane potential—dependent cell sorting.

In some ments, the at least one OSC is obtained from ovarian tissue. In other embodiments, the at least one OSC is obtained from a non-ovarian tissue.

In some embodiments, the non-ovarian tissue is blood. In other embodiments, the non- ovarian tissue is bone marrow.

In some embodiments, the composition comprising mitochondria obtained from at least one OSC or at least one progeny of an OSC, is the cytoplasm of the cells without a s. In other embodiments, the composition comprising mitochondria obtained from at least one OSC or at least one progeny of an OSC is a purified preparation of mitochondria.

In a related embodiment there is provided a method of isolating a population of functional mitochondria from at least one OSC, or at least one progeny of an OSC. The method ses the steps of incubating a ition comprising at least one OSC, or at least one progeny of an OSC, with a mitochondrial tracking probe under conditions sufficient to bind the probe to the functional mitochondria and sorting the functional mitochondria from the non-fimctional mitochondria, y isolating the population of fiinctional mitochondria from at least one OSC, or at least one progeny of an OSC. In some embodiments, non-functional mitochondria are excluded from the population of onal mitochondria.

In some embodiments, the mitochondrial tracking probe is a non-oxidation dependent probe. In some embodiments, the mitochondrial tracking probe is an accumulation dependent probe. In some embodiments, the mitochondrial tracking probe is a d oxidative state probe. In some embodiments, the sorting step includes fluorescence-activated cell sorting.

In another related embodiment there is provided a method of identifying a population of functional mitochondria obtained from at least one OSC, or at least one progeny of an OSC. The method comprises the steps of: a) incubating a composition comprising at least one OSC, or at least one progeny of an OSC, with a fluorescent reduced oxidative state probe and a fluorescent lation dependent probe under conditions sufficient to bind the fluorescent reduced oxidative state probe to functional mitochondria in the composition and bind the fluorescent accumulation dependent probe to total mitochondria in the composition; b) obtaining a composition comprising the functional mitochondria using cence-activated cell sorting, wherein the composition excludes non-functional mitochondria; c) determining the amount of functional mitochondria and the amount of total ondria; and d) calculating the ratio of functional mitochondria to total mitochondria; and c) ining whether the ratio is greater than about 0.02, thereby identifying a tion of functional mitochondria obtained from at least one OSC, or at least one progeny of an OSC.

In some embodiments, the cent accumulation dependent probe can fluoresce in one portion of the spectrum (e.g., green). In other embodiments, the cent reduced oxidative state probe can fluoresce in a ent portion of the spectrum (e.g., red).

In another related embodiment there is provided a composition comprising functional mitochondria obtained according to a method comprising the steps of: a) incubating a composition comprising at least one OSC, or at least one progeny of an OSC, with a cent reduced ive state probe and a cent accumulation dependent probe under conditions sufficient to bind the fluorescent reduced oxidative state probe to functional mitochondria in the ition and bind the cent accumulation dependent probe to total mitochondria in the composition; and b) obtaining a composition comprising the functional mitochondria using cence-activated cell sorting, wherein the composition excludes non-functional mitochondria.

In another related embodiment there is ed a kit sing a composition comprising isolated OSC mitochondria or mitochondria obtained from a progeny of an OSC and instructions for use. In one embodiment, the composition is at least about 85%, 90%, 95% free of cells or non- onal mitochondria.

In another related embodiment there is provided a kit sing at least one isolated mitochondrion obtained from an OSC or at least one progeny of an OSC and instructions for use.

Another aspect of the invention provides a method for increasing the ATP-generating capacity of an oocyte. The method comprises the steps of: a) providing a composition comprising mitochondria obtained from at least one OSC or at least one progeny of an OSC that is autologous to the oocyte, the at least one OSC having been isolated from ovarian tissue; and b) injecting the composition sing mitochondria into the oocyte in vitro, wherein the OSC is an isolated non-embryonic stem cell that is cally competent and expresses Vasa, Oct-4, Dazl, Stella and ally a stage specific embryonic antigen.

In some embodiments, the composition comprises 1x103 to 5x104 mitochondria.

In other embodiments, the OSC or progeny of an OSC produces at least 5-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In yet other embodiments, the OSC or progeny of an OSC produces at least 10-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In still other embodiments, the OSC or progeny of an OSC produces at least 50—fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. In certain, embodiments, the ovarian somatic cell or hymal stem cell is autologous.

In other embodiments, the OSC or progeny of an OSC produces at least lOO-fold more ATP per fg mtDNA than an ovarian somatic cell or hymal stem cell. In n embodiments, the ovarian somatic cell or mesenchymal stem cell is autologous.

In some embodiments, the oocyte is obtained from a human female of advanced maternal age. In other embodiments, the oocyte is obtained from a human female with low ovarian reserve.

In some embodiments, the composition comprises mitochondria that have been isolated by centrifugation. In other embodiments, the composition ses mitochondria that have been isolated by mitochondrial membrane ial-dependent cell sorting.

In some embodiments, the at least one OSC is obtained from ovarian . In other embodiments, the at least one OSC is obtained from a non-ovarian tissue.

In some embodiments, the non-ovarian tissue is blood. In other embodiments, the non-ovarian tissue is bone marrow.

In some embodiments, the composition comprising mitochondria obtained from at least one OSC or at least one progeny of an OSC, is the cytoplasm of the cells without a nucleus. In other embodiments, the composition sing mitochondria obtained from at least one OSC or at least one progeny of an OSC is a purified preparation of mitochondria.

In another related embodiment there is provided an oocyte prepared by a method comprising the steps of: a) obtaining a composition comprising mitochondria obtained from at least one OSC or at least one progeny of an OSC that is autologous to the oocyte; and b) injecting the composition of mitochondria into the oocyte.

In another related embodiment there is provided a composition of mitochondria obtained from at least one OSC or at least one progeny of an OSC, wherein the composition comprises a population of mitochondria in which greater than about 75%, 85%, 90%, or about 99% ofthe ondria are high ATP-generating ty mitochondria.

In still r related embodiment there is provided compositions comprising a population of mitochondria in which less than about 5% to about 25% of the mtDNA comprises a deletion mutation within nucleotides 8470-13447 of the mitochondrial genome, and methods pertaining to such compositions.

Any discussion of documents, acts, als, s, es or the like which has been included in this specification is solely for the purpose of providing a t for the t invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in New Zealand or elsewhere before the priority date of this application.

Other features and ages of the invention will be apparent from the detailed description, and from the claims. Thus, other aspects of the invention are described in the following disclosure and are within the ambit of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS The following Detailed Description, given by way of example, but not intended to limit the invention to specific embodiments described, may be understood in ction with the accompanying figures, incorporated herein by reference.

Figure 1 s validation of a fluorescence-activated cell sorting (FACS)—based protocol for OSC isolation. In Figure la, immunofluorescence analysis ofVASA expression (with DAPI counterstain) is shown in adult mouse ovaries using antibodies against the NI-Iz or COOH terminus ofVASA (scale bars, 50 mm). In Figure lb, immunomagnetic sorting of dispersed mouse s or isolated oocytes is shown using antibodies t the NH; or COOH terminus ofVASA. Fraction 1 contains cells plus beads prior to separation, Fraction 2 is a wash or flow-through fraction (non-immunoreactive) and Fraction 3 is a bead fraction (VASApositive . In Figure 1c, FACS analysis of live or permeabilized cells from dispersed mouse ovaries using antibodies against the NHZ or COOH terminus ofVASA is shown. Viable sitive cells are only detected with the COOH antibody (dashed box) whereas permeabilization enables isolation of VASA-positive cells using the NI-Iz antibody (dashed box). In Figure 1d, permeabilization of viable ositive cells (dashed box) obtained with the COOH antibody enables lation ofthe same cells by FACS using the NH: antibody (dashed box). In Figure lo, a schematic representation ofthe FACS protocols employed using the VASA-COOH dy for ion ofviable OSCs is shown. Figure 1fdepicts gene expression analysis of germlinemarkers [Blirnpl (alsoreferredtoasPRdomaincontaining l wichNFdomainor , Stella, Fragilis (also referred to as interferon d transmembrane protein 3 W), Tert (telemerasereverse transcriptase), Vasa, Dazl (deleted in azoospermia like)] and oocyte marl-era [Nobox (newborn ovary homeobox), Zp3 (zona pellucida glycoprotein 3), GdiD (growth diiferentiation factor 9)] in each cell fiaction produced during the ovarian dispersion process to obtain cells for FACS-based isolation ofOSCs using the VASA-COOH antibody (+ve, VASA—posifive viable cell fi'action after FACS; —ve, VASA-negative viable cell fraction alter FACS; No RT, PCR ofRNA sample without reverse transcription; B—actin, sample loading control).

Figure 2 depicts OSC fractions isolated fi'om adult mouse ovaries by imnumomagnetic bead g that contain contaminating oocytes. Gene ermression analysis ofgermline markers (Blimpl, Stella, Fragilis, Tert, Vssa, Dazl) and oocyte-specific markers (Nobox, Zp3, G619) is shaminyoung adultmouseovaries (positivecontrol) orthe final cell fractionobtained following VASA-COOH antibody-based immrmomagnefic bead sorting of dispersed young adult mouse ovaries (No RT. PCR ofsorted cell RNA sample without reverse transcription; 6- actin, sample loading control).

Figure 3 depicts isolation ofVASA-positive cells fi'om adult mouse and human ovaries using FACS. In Figure 3a and b, the representative histological appearance ofadult ovarian tissue used forhuman (a) and mouse (b) 08C isolation is shown. Scale hm. 100 run. In Figures 3c and d, the morphology ofviable cells isolated by FACS based on cell-surface expression of VASA is shown. Scalebars, 10 um. Figure idesthc gencmlpressionprofile ofstarting ovarian material and fieshly—isolated 0805, showing assent of 3 different patients as examples for human tissue analysis (No RT: PCR ofRNA sample Without reverse ription; fl—aetin, sample loading control). In Figure ugh Figure 31:, a temtoma formation assay g an absence oftumors in mice 24 weeks after receiving injections ofmouse 0808 (31) compared with development rs inmice 3 weeks after injection ofmouse embryonic stem cells (ESCs) is shown (Figure 3g through Figure 35; panels 3h through 3j show examples ofcells fi'omall three yers, withnem'alrosettehighlighmdinpanelah, inset), alongwitha summary ofthe experimental es (3k).

Figure 4 s firnetional eggs obtained from mouse 0808 silver intraovarian transplantation. In Figures 4a and 4b, examples ofgrowing follicles containing gative and GFP-positive (hematoxylin rstain) oocytes are shown in ovaries ofwild-type mice injected with GFP-expressing 080: 5-6 months earlier. In Figure 4e, examples ofovulated GFP-negative eggs (in s-oocyte xes), and resultant embryos [2-cell, 4-cell, compact morals (CM) and early blastocyst (EB) stage s are shown as examples] generated by IVF are shown, ing induced ovulation ofwild-type female mice that received intreovarian transplantation of GFP-expressing OSCs 5-6 months earlier. In Figures 4d and 4e, examples of OPP-positive eggs (in ctnntflus-oocyte complexes) obtained from the oviducts are shown following induced ovulation ofwild-type female mice that resettled intraovarian transplantation of GFP-expressing OSCs 5-6 months earlier. These eggs were in vitro fertilized using wild-type sperm, resulting in z-cell embryos that progressed fluough prennplantatian development [examples ofGFP-positive embryos at the 2-cell, 4-cell, 8-oell, compacted morula (CM), expanded morula (EM), blastocyst (B) and hatching blastocyst (H13) stage are shown] to form hatching blastocysts 5-6 days after fertilization.

Figure 5 depicts germ cell colony formationbymouse and human OSCs in vitro. quutmofluoreseence-based analysis ofVASA expression is shown in Figures 51) and 5d; (with DAPI rstain) in typical germ cell colonies formed by mouse (5a, 5b) and hmnan (5c, 5d) 0808 after establishment on mouse nic fibroblasts (MEFs) in vitra al colonies highlighted by white dashed lines).

Figtn'e 6 depicts evaluation ofmouse and human ovary-derived VASA-positive cells in defined culttues. Figures 6a tln-ough 6d show assessment ofOSC proliferation by dual detection ofVASA sion and BrdU incorporation in mouse (6a, 6b) and human (6c, 6d) OSCs maintained in MEF-free cultures. Figure 6c shows the typical growth curve -fi-ee cultures ofmouse OSCs afier passage and seeding 2.5 X 10" cells per well in 24-well culture plates. Figure 6f shows FACS analysis using the COOH antibody to detect cell-sm'fsce expression ofVASA in mouse OSCs ailm- months agation (example shown, passage 45).

Figure 63 indicates the gene expression profile ofstarting ovarian material and canned mouse and human OSCs afier 4 ormore months ofpropagation in vitro (No RT, PCR ofRNA sample without reverse transcription; fl-actin, sample loading control). Two different human OSC lines (0801 and OSCZ) ished from two different ts are shown as examples. Figure 611 and 6i show representative imnmnofluorescence analysis ofBLlMPl, STELLA and FRAGIIJS sion in mouse (11) and human (i) OSCs in MEF-fi'ee cultures. Cells were cormteratained with DAPI and rhodamine-phalloidin to visualize nuclearDNA and asmic F-actin, respectively.

Figure 7 depicts spontaneous oogenesis from cultured mouse and human 0803. Figures 7a through 7c provide examples ofimmature ooeytes formed by mouse OSCs in culture, assessed by morphology (7a), expression of oocyte marker proteins VASA and KIT (7b; note cytoplasmic localization ofVASA), and the presence ofmRNAs encoding the oocyte marker genes Vase, Kit, Msy2 (also ed to as YboxproteinZ orbe2), Nobox, th8, Gd19, Zpl, Zp2 and Zp3 (7c; No RT: PCR ofRNA sample without reverse transcription; n, sample loading control). Scale bars, 25 pm. Figure 7d indicates the number ofimmature oocytes formed by mouse OSCs 24, 48 and ’72 hours after passage and seeding 2.5 X 104 cells per well in 24- well culture plates (culture atants were ted at each time point for determination, thus the values meatnumbers ted over each 24 hour block, not cumulative numbers; mean i SEM, n= 3 independent cultures). Figures 7e through 7g show in vitro oogenesis fi'orn human OSCs, with examples ofimmatru'e oocytes formed byhuman 030s in culture (71; morphology; 7g, expression ofoocyte marker proteins VASA, KIT. MSY2 and EDIE) and numbers fonmd following e and swding of 2.5 x 10" cells per well in 24-well culture plates (7e; mean :1: SEM, n = 3 independent cultures) shown. The presence ofmRNAs encoding oocyte marker germs (Vasa, Kit, Msyz, Nobox, thB, Gdf9, Zpl, Zp2, Zp3) in human OSC- derived oocytes is shown in panel c along with results for mouse OSC-derived oocytes. Scale bars, 25 pm. In Figure 7b, innnunofluorescence-based detection of the c recombination markers, DMCl (dosage suppressor ofmold homolog) and SYCP3 (synaptonemal complex protein 3) (DAPI cormterstain), is shown in nuclei ofcultured human OSCs; human ovarian stromal cells served as a negative control. In Figure 7i, FACS-basad ploidy analysis ofcultured human OSCs is shown 72 hours after passage. Results from ploidy analysis ofcultured human fibroblasts (negative control) and cultured mouse OSCI are ted in Figure 9.

Figure 8 depicts the detection ofoocyte-specific markers in adult human ovaries.

Imrnlmofluorescence analysis ofVASA (8a), KIT (8b), MSYZ (8c) and LHX8 (8d,) expression in oocytes in adult human ovarian cortical tissue is shown (see also Figure 10h). Sections were cormterstained with DAPI for visualization ofnuclei. Scale bars, 25 Figure 9 depicts ploidy is ofhuman fibroblasts and mouse 080s in culture. Figure 9a and 9b show representative FACS-based assessment ofploidy status in cultures ofactively- dividing human fetal kidney fibroblasts (9a) and in mouse OSCs collected 48 hours alter (9b). Haploid (In) cells were only detected in the germline cultlnes, consistent with results from analysis ofhuman OSCs maintained in vitm (see Figure 7i), whereas all culhnes contained diploid (2n) and tetraploid (4n) populations of cells.

Figure 10 s generation of oocytes from human 080s in human issue. Direct (live-cell) GFP fluorescence analysis ofhuman ovarian cortical tissue following dispersion, ation with GFP-hOSCs (10a) and in vitro culture for 24-72 hours (10b. 10c) is shown.

Note the formation of large single GFP-positive cells surrounded by smaller gative cells in compact structures resembling follicles (Figures 10b and 10c; scale bars, 50 pm).

Examples of immature follicles containing GFP-positive oocytes (highlighted by black arrowheads, against a hernatloxylin rstain) in adult human ovarian cortical tissue ed with GFP-hOSCs and xenograflled into NOD/SCID female mice are shown e 10d. 1 week post-transplant; Figure 10f, 2 weeks post-transplant). Note comparable follicles with GFP-negative oocytes in the same grafls. As ve controls, all immature les in human ovarian cortical tissue prior to GFP-hOSC injection and xenografting (10c) or that ed vehicle injection (no GFP-hOSCs) prior to xenografiing (10g) contained GFP-negative oocytes after processing for GFP detection in parallel with the samples shown above. Figure 10h shows dual immunofluorescence analysis of GFP expression and either the diplotene stage oocyte-specific marker MSY2 or the oocyte transcription factor L}D(8 in xenografls receiving GFP-hOSC injections. Note that GFP was not detected in grafts prior to SC injection, whereas MSY2 and LHXS were detected in all s. Sections were counterstained with DAPI for visualization of nuclei. Scale bars, 25 um.

Figure 11 s morphometry—based assessment of oocyte formation in human ovarian xenografts following GFP-hOSC transplantation. The total number of primordial and primary follicles in 3 randomly ed human ovarian cortical tissue s (labeled 1, 2 and 3) are shown, 7 days after injecting GFP-hOSCs and xenografiing into NOD/SCID mice, which contain GFP-negative (host-derived) or GFP-positive (OSC-derived) oocytes (see Figures 10d through 10g for examples).

Figure 12 depicts eservation and thawing of human ovarian cortical tissue and freshly-isolated human OSCs. Figure 12a and 12b show the histological ance of adult human n cortical tissue before and after cation, highlighting the maintenance of tissue integrity and the large numbers of oocytes (black arrowheads) that survive the freeze- thaw procedure. In Figure 12c, the percent cell loss following freeze-thaw of freshly-isolated human OSCs is shown (results from two different patients).

Figure 13 depicts an overview of an ous Qermline Mitochondrial E_nergy Iransfer (AUGMENT) procedure. Note that OSCs used as a source of mitochondria for the transfer, and the egg to be fertilized which will receive the OSC mitochondria, are obtained from the same subject.

Figure 14 depicts mitochondrial staining with MitoTracker Green FM (Invitrogen M7514, Life Technologies Corp., Carlsbad, CA) in cultured human ovarian somatic cells and cultured human OSCs obtained from the same patient.

Figure 15 depicts PCR analysis of the 4977-bp deletion in mtDNA from cultured OSCs and t matched ovarian somatic cells.

Figure 16 depicts the results of an ATP assay.

Figure 17 depicts FACS—based germ cell purification or isolation fiom bone marrow mononuclear preparations of adult female mice during estrus of the female reproductive cycle using cell surface expression ofVasa to isolate the cells.

Figure 18 depicts FACS—based germ cell purification or isolation fi'om peripheral blood mononuclear cell preparations of adult female mice during estrus of the female reproductive cycle using cell surface expression ofVasa to e the cells.

Figure 19 depicts mitochondria following staining with mitotracker M7514 and cell lysis. Human OSCs were incubated with M7514, and then lysed to release the d mitochondria using c shock. The entire population (mitochondria from lysed cells and residual unlysed stained cells) was analyzed by FACS. The lefl: panel shows mitochondria from lysed cells, which are easily distinguishable from mitochondria contained in al unlysed cells based on size (forward scatter; . Fluorescence intensity (FITC-A) revealed two distinct populations of mitochondria from lysed cells, one having high intensity (Mito MT high), and one having low intensity (Mito MT Low). Functional mitochondria are known to have a greater uptake and ion ofthe stain, and thus fluoresce at a higher intensity (Invitrogen technical staff, Life Technologies Corp., Carlsbad, CA).

Figure 20 s the kinetics of ATP production capacity by mitochondria isolated from different human cell types.

Figure 21 depicts ATP production capacity over 10 minutes by mitochondria isolated from ent human cell types.

Figure 22 s mtDNA deletion analysis in human mesenchymal stem cells and human ovarian soma.

DETAILED DESCRIPTION OF THE INVENTION Definitions Unless otherwise defined, all cal and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions will control.

“Oogonial stem cells” (OSCs), also known as female germline stem cells, are derived from postnatal sources and express markers ing Vasa, Oct-4, Dazl, Stella and optionally an SSEA. 0805 are mitotically competent (i.e., capable of mitosis) and do not express oocyte markers including growth/differentiation factor-9 ("GDF—9"), and zona pellucida glycoproteins (e.g., zona pellucida glycoprotein-3, "ZP3"), or markers of meiotic recombination such as synaptonemal complex protein-3 ("SYCP3" or ). OSCs can be obtained from the postnatal ovary. OSCs are known in the art and are described in U.S. Patent No. 7,955,846, the entire contents of which are incorporated herein by reference. OSCs are additionally described by Johnson et al., Nature 428:145—150; Johnson et al., Cell 2005 122:303—315; Wang et al., Cell Cycle 2010 9:339—349; Niikura et al., Aging 2010 2:999—1003; Tilly et al., Biol Reprod 2009 802—12, Tilly et al., Mol Hum Reprod 2009 15:393—398; Zou et al., Nat Cell Biol 2009 11:631—636; Pacohiarotti et al., Dw'erentiatian 2010 79: ). the contents ofwhich are incorporated herein by reference. ably, fire osc offlre invenfion is a hmnan osc. herein,the“progeny C’referstc all daughtercells derivedfromOSCs ofthe invention, including progenitor cells and differentiated cells that in or achieve oogenic potential (i.e.I the ability to form an oocyte) and functional mitochondria. Preferably, the OSC progeny ofthe invention is a human OSC progeny.

As used hereim the term "functional mitochondria” refers to mitochondria that produce ATP and can he used interchangeably with the term ring mitochondria.” OSCs my additionally be obtained from the bone marrow, peripheral blood or mnbilical cord blood. Bone marrow derived OSCs ofthe invention can also circulate throughout the body and most preferably can be localized in bone marrow. peripheral blood and ovary.

Bone marrow derived OSCs express markers including Oct 4, Vase, Dazl, Stella, Fragilis, and optionally Nohox, Kit and Sea-l. Bone marrow derived OSCs are mitotically competent (i.e., e ofmitosis) and do not express GDP-9, zona pellucida proteins (e.g., ZPB) or SCPB. For additional s on bone marrow-derived OSCs, see, U.S. Patent Pub. No. 20060010509, the entire contents of which are incorporated herein by reference for their description of080s in bone marrow. For additional details on peripheral blood and umbilical cord blood derived OSCs, see, U.S. Patent Pub. No. 2006001596], the entire contents ofwhich are incorporated herein by reference for their description ofOSCs in the peripheral blood.

Oct-4, also referred to as POU domain class 5 transcription factor 1 or Pousfl, is a gene expressed in female germline stem cells and their progenitor cells. The Oct-4 gene encodes a transcription factor that is involved in the establishment ofthe mammalian germline and plays a significant role in early germ cell specification (reviewed in Scholer,m Genet. 1991 mot-323.329). In the developing mammalian embryo, Oct-4 is egulated during the differentiation ofthe epihlast, ally ng d to the germ cell lineage. In the germline, Oct-4 expression is ted separately from epiblsst wrpressicn. Expression ofOct-4 is a phenotypic marker oftotipotency (Ycorn et a1., Development 1996 122:881-888).

Stella, also commonly referred to as developmental plm'ipctency associated 3 or Dppa3, is a gene sed in female germline stemcells and their progenitor cells. Stella is a novel gene specifically expressed in primordial germ cells and their descendants, ing oocytes (Bortvin et al., BMCDeveIOpmental Biology 2004 4(2):1-5). Stella encodes a protein with a SAP—like domain and a splicing factor motif-like structure. Embryos deficient in Stella expression are compromised in preimplantaticn dewlopment and rarely reach the blastocyst stage. Thus, Stella is a maternal factor implicamd in early embryogenesis.

Deal is a gene expressed in female germline stem cells and their progenitor cells. The autosomal gene Dazl is a member ofa family ofgenes that contain a consensus RNA binding domain and are expressed in germ cells. Loss of expression of an intact Dazl protein in mice is associated with failure of germ cells to complete meiotic prophase. Specifically, in female mice null for Dazl, loss of germ cells occurs during fetal life at a time coincident with progression of germ cells through meiotic prophase. In male mice null for Dazl, germ cells were unable to progress beyond the leptotene stage of meiotic prophase 1. Thus, in the absence of Dazl, progression through meiotic prophase is interrupted (Saunders et al., Reproduction 2003 126:589-597).

Vasa, also referred to as DEAD box polypeptide 4 (“DEAD” disclosed as SEQ ID No: 1) or Ddx4, is a gene sed in female germline stem cells and their progenitor cells. Vasa is a component of the germplasm that encodes a DEAD-family (“DEAD” disclosed as SEQ ID No: 1) ATP-dependent RNA helicase (Liang et al., Development 1994 120: 1201 -121 1; Lasko et al., Nature 1988 335:611-167). The molecular fimction of Vasa is directed to binding target mRNAs involved in germ cell establishment (e.g., Oskar and , oogenesis, (e.g., Gruken), and translation onset (Gavis et al., Development 1996 1101521- 528). Vasa is required for pole cell formation and is exclusively cted to the germ cell lineage throughout development. Thus, Vasa is a molecular marker for the germ cell lineage in most animal species aki et al., Cell Structure and Function 2001 26:131-136).

Stage-Specific Embryonic Antigens are optionally expressed in female ne stem cells and expressed in female germline stem cell progenitors of the invention. Stage- Specific Embryonic Antigen—1 (SSEA—l) is a cell surface embryonic antigen whose functions are associated with cell adhesion, migration and entiation. During hypoblast formation, SSEA-l positive cells can be identified in the blastocoel and hypoblast and later in the germinal crescent. SSEA-l functions in the early germ cell and neural cell development. (D'Costa et al., Int J. Dev. Biol. 1999 43(4):349-356; Henderson et al., Stem Cells 2002 —337). In specific embodiments, expression of SSEAs in female ne stem cells may arise as the cells differentiate. SSEAs useful in the invention include SSEA—l, -2, -3, and -4.

The term "autologous" as used herein refers to biological compositions obtained from the same subject. In one embodiment, the biological composition es OSCs, OSC- derived compositions and oocytes (i.e., mature oocytes). Accordingly, in conducting methods of the invention, the female germ cell cytoplasm or mitochondria used for transfer and the recipient oocyte into which the aforementioned compositions are transferred are ed from the same t.

The term "isolated" as used herein refers to an OSC, mitochondrion or ition derived from an OSC (e.g., cytoplasm, mitochondrial preparation), which has been physically separated or removed from its natural biological environment. An ed OSC, mitochondrion or composition need not be purified.

The term "exogenous" as used herein refers to transferred cellular material (e.g., mitochondria) that is removed from one cell and transferred into r cell. For example, OSC derived mitochondria that have been transferred into an oocyte, even if both are derived from the same subject, would be exogenous.

A "subject" is any live-bearing member of the class mamrnalia, including humans, domestic and farm animals, and zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle and higher primates.

As used herein, the term "advanced maternal age" as it s to humans refers to a woman who is 34 years of age or older. As used herein, the term "oocyte—related infertility" as it relates to humans refers to an inability to ve after one year of unprotected intercourse which is not caused by an anatomical abnormality (e.g., blocked oviduct) or pathological condition (e.g., uterine fibroids, severe endomettiosis, Type II diabetes, stic ovarian e).

As used herein, the term "low ovarian reServe" as it relates to humans refers to a woman who exhibits a circulating Follicle Stimulating Hormone 0:SH) level greater than 15 miu/ml in a "day 3 FSH test," as described in Scott et al., Fertility and Sterility, 1989 51:651-4, or a circulating Anti-Mullerian Hormone (AMH) level less than 0.6 ng/ml, or an antral le count less than 7 as measured by ultrasound.

In this disclosure, "comprises," "comprising," “containing" and "having" and the like will be understood to imply the inclusion of a stated element, integer or step, or group of ts integers or steps, but not the ion of any other element, integer or step, or group of elements, integers or steps. "Consisting essentially of" or "consists essentially" can likewise have the meaning ascribed in US. Patent law and the term is open—ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence ofmore than that which is recited, but excludes prior art embodiments.

The term ed" or "reduce" or "decrease" as used herein generally means a decrease of at least 5%, for example a decrease by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and ing a 100% decrease (i.e. substantially absent or below levels of detection), or any decrease between 5-100% as compared to a nce level, as that term is defined herein, and as determined by a method that achieves statistical significance (p <0.05).

The term "increase" as used herein generally means an increase of at least 5%, for example an increase by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase (i.e. substantially above levels of detection), or any increase n 5-100% as compared to a reference level, as that term is defined herein, and as determined by a method that achieVBs statistical significance <0.05).

As used herein “an increase in ATP generation or production" refers to an amount of ATP production that is at least about 1-fold more than (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, , 30, 40, 50, 60, 70, 80, 90, 100, 1000, 10,000-fold or more) the ammmt ofATP production areference level, asthattermis definedherein. A'I‘Pproduetion canbemeasluedbystandard s known in the art.

As used herein, "high ATP-generating capacity mitochondria" refers to mitochondria having a high mitochondrial membrane potential, as determined by a probe which can- distinguish between high and low (or between high and medium/low) membrane ial. One method ofidentifying mitochondria with high mitochondrial membrane potential is the use of the fluorescent probe 5,5',6,6'-teuaohloro-l,1',3,3'-tetraediylbenzimidazolyl carbooyanine iodide (JC-l , ogen T3168, Life Technologies Corp., Carlsbad, CA). which fluoresces red-orange (590 run) in high quality mitochondria but fluoresces geen (510-520 um) in medium and/or low y mitochondria. (See, e.g., Gamer et a1., Bio. Reprod. 1997 57:1401-1406; Reers et al., Biochemistry 1991 30:4480-4486; Cossariza et a1, Biochem Biophys Res Commun 1993 197:40- 45; Smiley et al., Proc NatlAcad SciUSA 1991 88:3671-3675).

As used herein, the term “standard” or 'refermce" refers to a measured biological parameter including but not limited to defects such as aneuploidy, mutation, chromosomal misalignrnent, meiotic spindle abnormalities, and/or mitochondrial dysfimction (aggregation, impaired ATP production), or the reduction or elimination of such defects, in a known sample against which another sample is ed; alternatively, a rd can simply be a reference number that represents an amount ofthe measured ical parameter that defines a baseline forcemparison. Thereference number canbe dcrivedfromeifllera sampletakenfi’oman individual, or a ity of individuals or cells obtained therefrom (e.g., oocytes, OSCs). That the “standard" does not need to be a sample that is tested, but can be an accepted refer-mes number or value. A series of standards can be developed that take into account an individual’s status, e.g., with t to age, gender, , height, ethnic background etc. A standard level can be obtained for example from a known sample from a different dual (e.g., not the individual being med). A known sample can also be obtained by pooling samples item a plurality viduals (or cells obtained flierefi’om) to produce a standard over an averaged pepulation. onally, a standard can be synthesized such that a series of standards are used to quantify the biological parameter in an individual's sample. A sample item the individual be tested canbeobtained atanearliertimepoint (preslnnablypriorto the onsetoftreannent) and serve as astanrlardorreferenceeomparedto asamplemkenfromthesame individualafierthe onset oftreatment. In such instances, the rd can provide a measure ofthe efficacy treatment. In specific embodiments, a “standard” or “reference” is an ovarian somatic cell (e.g., an aged-matched ovarian somatic cell obtained fiom a female subject having a na] reproductive system) or an aged-matched mesenchymal sum cell.

Rangesprofidedhereinareunderstoodtobeshorthandfor allofthevalueswithinflie range. For example, a range of l to 50 is understood to include any number, combination of numbers, or sub-range Earn the group mean, 1, 2, 3, 4, 5, 6, 7, 8,9,10, 11, 12. 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Other ions appear in context throughout this disclosure.

Compositions and Methods of the Invention Isolation of OSC: Adult ovarian cortical tissue can be obtained using a minor laparoscopic procedure known in the art to collect a small (e.g., 3x3x1 mm) ovarian cortical biopsy, which is then processed for OSC isolation. See Gook et al., Human Reproduction, 2004 20(1):?2-78.

Isolation n OSCs fi'om adult ovarian cortical tissue can be performed described in Example 1, Figure l or as previously described in the art, or using comparable teclmiques. See, for example, paragraph 01 16_ofU.S. Patent Pub. No. 20060010508, and Zen et al., Nature Cell Bioiagy 2009 5:631-6. Bpub 2009 Apr 12. OSCs can also be obtained from non- cvarian s. such as bone nun-ow or eral blood. Bone marrow and peripheral blood derived OSCs canbe isolatedbystandardmeans knowninflreartforthe separation cella from, for example, the marrow or blood (e.g., cell sorting). Optionally, the isolation protocol includes tion ofa in~ motion that is ed ofhematopoietic cells. Additional selection means based on the characwristic profile ofgene expression in OSCs (e.g., Vase, Oct- 4, Dazl, Stella, Fragilis) can be employed to further purify or isolate the desired population of cells and to reduce or ate other cells and material from the biological sample from which they were obtained (e.g. bone marrow, eral blood). For exanmle, the methods dwm’bed in Example 1. Figure 1b have been applied to a mononuclear fraction ofblood cells and bone marrow cells to obtain purified or isolated OSCs from non-ovarian sources. Briefly, cells were incubated with a rabbit anti-VASA antibody (ab13840; Abcam, Cambridge, MA) for 2.0 minutes, wamd, and incubated with goat anti-rabbit IgG conjugebd to allophcocyanin (APC) for 20 minutes, and washed again. Labeled cells in the eluate were isolated by fluorescence- activated cell sorting (FACS) using a BD Biosciences FACSAria It cytometer (Harvard Stem Cell Imtitute, Boston, MA), gated against negative (unstained and no y antibody) controls. Propidium iodide was added to the cell sionjust prior to setting for dead cell exclusion. s obtained using cell sin-face expression ofVase to isolate OSCs from non- ovarian s are provided in Figures 17 and 18, where the FACS-based germ cell purificatioa ofbone marrow and peripheral blood preparations fiorn adult female mice during estrus ofthe female reproductive cycle is shown.

Preparation ofOSC Derived Compositions and Methods of Transfer Methods for the preparation and transfer ofmitochondria are known in the art and be carried out-as previously described in the art, or using comparable techniques.

See, for example, Perez et al.. Cell Death andDrfi'er-entiation 2007 3:5?44-33. Epub 2006 Oct 13, and Perez et al., Nature 2000, 403:500-1, the contents each ofwhich are incorporated herein reference. Briefly, OSCs can be isolated and ed as described above. In one method, when OSC cultm'es reach 80% confluency, 2 ml ofmitochondrial lysis buffer (0.3 M sucrose, 1 mM EDTA, 5 mM MOPS, 5 mM KH2P04, 0.1% BSA) is added to each plate, and the cells are removedusingacel] soraper.'I‘hecell suspensionis erredintoasmall glasstissuedouncer and homogenized until smooth (approximately 10 up-and—down Strokes), and the lysate centrifuged at 600 g for 30 minutes at 4°C. The aupernatantis removed and spun at 10,000 g for 12 s at 4°C, and the ing crude mitochondrial pellet is resuspended in 0.2 ml of 0.25 M sucrose. This sample is then layered over a 25—60% Percoll y gradient dilumd with 0.25 M sucrose and centrifuged at 40,000 g for 20 minutes at 17°C. The interfacebandis extracted from the gradient and washed in 2 volumes M sucrose before a final centrifiigation at 14,000 g for 10 min at 4°C to yield a mitochondrial pellet.

The mitochondrial pellet can also be ed as described Frezza et al. Nature Protocols 2007 2:287-295, the contents ofwhich are incorporated herein by reference. c embodiments ofthe invention, the total OSC-derived mitochondrial population in tissue, cell, lysed cell, or on thereofcan be isolated, characterized and/or ated using a FACS-based method with a fluorescent probe that specifically binds to mitochondria in mitochondrial membrane potential (MMP)-indepmdent warmer. Fluorescent probes that specifically bind to mitochondria in a MED-independent manner include, but are not limited accumulation dependent probes (e.g., 101 (red spectrum; Invitrogen T3168, Life Technologies Corp., Carlsbad, CA), lldito'l‘raeker Deep Red FM rogen M22426, Life Technologies Corp., Carlsbad, CA) and JC-l (green spectrum; Invitrogen T3168, Life Technologies Corp., ad, CA). Functional (e.g., respiring) mitochondria can be sormd and collected, preferably with exclusion ofresidual unlysed cells and non-fimctional mitochondria, based on size and fluorescence intensity using mitochondrial ng probes that indicate mitochondrial mass including, but not limited to, non-oxidation dependent probes (e.g., MitoTracker Green FM (Invitrogen M7514, Life Technologies Corp., Carlsbad, CA). Details ofan exemplary protocol for conducting FACS with a non-oxidation ent probe are provided below in Example 9.

Optionally, the FACS-based method can also be employed to selecu'vely yield a substantially pure population of functional (c.g., respiring) mitochondria using a mitochondrial membrane cent probe that specifically binds to mitochondria in a LAMP-dependent manner.

Fluorescent probes that specifically bind to mitochondria in a WIMP-dependent manner include, but are not limited to, d oxidative state mitou-acker probes (e.g., MitoTracker Red CM- HZXRos (Invitrogen M7513, Life Technologies Corp, Carlsbad, CA) and MitoTracker Orange MRos (Invitrogen M7511, Life Technologies Corp., Carlsbad, CA). Fln'thermore, dual- labeling using LIMP-dependent and HEMP-independent probes can be ted to quantitate the ratio of fimctional to total mitochondria in a tissue, cell, lysed cell or fiaction derived therefi’om. In specific embodiments, the ratio is greater than about 0.02, 0.025, 0.033, 0.04, 0.05, 0.1, or about 0.2. When using probes for differential screening based on MMP, spectral coloris themajordcterminmg factortodcsignate functional ondria, andforward scatter can be used to distinguish the fluorescent mitochondria released fi'om lysed cells fi'om those still contained in residual unlysed cells.

Mtochondrlal pellets can also be prepared as described by Taylor et al., Nat. Biotechnol. 2003 21(3): 239-40; Hanson et al., Electrophoresis. 2001 22(5): 950-9; and Hanson et al., J.

Biol. Chem. 2001 276(19): 16296-301. In specific embodiments of the invention, the total OSC- derived mitochondrial population in a tissue, cell, lysed cell, or fi'action f can be isolated, characterized and/or enumerated using a difl‘erential ccntrifugation method such as that described herein at Example 10 or using a sucrose gradient separation procedure such as that described herein atExample 11.

Following ion, ment ofmitochondrial function or mtDNA integrity (e.g., mutations and ons) can be conducted ing to methods known in the art (Duran et al., Fertility and Sterilin 2011 96(2):384-388; Aral et a1., Genetics and Molecular Biology 2010 33:1-4; Chan et al., Molecrdar Human Reproduction 2005 11(12):843-846; Chen et al., BMC Medical Genetics 2011 12:8 and Example 8). tions ofmitochondria sorted according to functional parameters (e.g., MMP dependent/active or MAP-independent/acfive plus inactive) or mitochondria from less preferred OSC soInces, including samples oflimited size, can be now be obtained according to the methods ofthe invention. Mitochondrial itions ofthe invention can generate, for example, about 1 pmol ATP per fg mtDNA to about 6 pmol A'IP per fgmtDNA (e.g., about 1, 2, 3, 4, 5, or 6 pmol ATP per fg mtDNA). In specific embodiments, between about 1.0 pmol to 1.4 pmol ATP per fg mtDNA is generated within about minutes to about 15 minutes.

The percentage tions in a population ofmitochondria can be assessed by first ining the number ofmitochondria present in a biological sample and next, ining the copy number ofmitochondrial DNA present in the sample. Standard mutation analysis can be employed and compared to the number chondria and capy number of mitochondrial DNA to ate the percentage of mutations in the population of mitochondria. For example, compositions and methods ofthe invention can provide a population ofmitochondria in which less than about 5% to about 25% (e.g., about 5%, 10%, 15%. 20% to about 25%) ofthe mitochondrial DNA comprises a deletion mutation within nucleotides 8470-13447 ofthe ondrial genome.

The material to be injecwd (e.g., mitochondrial suspension) is transferred to microinjection needle according to methods known in the art. Mioromjection needles and holding es can be made using a Sutter puller (Sum Instwmssnts, Novato, CA, USA) and a De Fonb'rune Mieroforge (EB Sciences. East Granby, CT, USA). The njecfion needles have inner diameters of 5 pm with blunt tips. The material to be injected is aspirated into the needle by negative n. Between about 1110’ — to about 51:104 mitochondria from OSCs their progenycan be ed (c.g., about 1, 2, 3, 4, 5, 6, 7, 8 to 9 x 10’; about 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 to about 5 x 10‘mitochondria). The mitochondrial suspension in sucrose (e.g., 5—7 p1 containing approximately 13:103 - 5x104 mitochondria fi'om OSCs or their progeny) can be injected into ooeytes using a Piece micromanipulator. Oocytes that survive the microinjection ure are transferred for culture and optionally. assessment or eservation prior to in vitro fertih'mflon or intrauterine insemination. Methods ofoccyte cryopreservation are well known in the art. For details, see for example, Porcu et al., Molecular and Cellular Endocrinology 2000 159:33—37; Mandelbaum, Human Reproduction 2000 15;43-47; and Fabbri et al., Molecular and Cellular Endocrinology 2000 169:39-42, the contents ofwhich are incorporated herein by reference.

Methods for the preparation and transfer ofnuclear-flee cytoplasmic fractions known in the art and can be carried out as previously described. See, for example, Cohen et a1., MolHumReprod 1998 4:269—80, the contents h are incorporated herein by nce.

Briefly, in one method, apprmdmately 4 hours afier egg retrieval, recipient eggs are exposed to 0.1% hyaluronidase, and mature eggs are selected for injection. All corona cells are removed with fine bore pipettes. Ooplasmic transfer can be performed by electrofusion ofOSC ooplaals with intact MII oocytes. Afier exposure to 0.1% hyaluronidase, muse are opened mechanically ushigamicrospear. OSCs are exposedtohI-I'I'FmediumcontainingcytochalosinB (CCB; Sigma Chemical Co., St Louis, MO, USA) for 10 min at 37°C. Partitioning ofhmnan MI] oocytes involves le cytochalasin B concentration depending on their sensitivity (~25 mglml). Ooplasts ous sizes are separated fi'orn OSCs by withdrawing a portion ofthe ooplssm enclosed in the plasma ne. ent and elecuofiision in a mannitol solution is performed after insertion of the OSC derived ooplast into the pcrivitelline space ofthe recipient egg from which the polar body was removed. This can be done with a wide-bored polished microtool ~30-‘-40 Inn in diameter. The ooplast is sucked into the microtool and released once the tool is placed deeply into the perivitelline space. Oocytes that survive the electrofusion procedure are transferred for culture and optionally, assessment or eservation prior to in vitro fertilization or intrauterine insemination.

Alternatively, conventional intracytoplasmic sperm injectiOn (ICSI) s can be employed in connection with the transfer ofnuclear-fies cytoplasmic fi'actions or isolated mitochondria. See, for example, Cohen et al., MolHum Reprod 1998 4:269—80, the contents of reineorporatedhereinbyreference.Asoneexample,thezonae oftherecipienteggs are opened ically over the polar body area using a microspear. The polar body is removed aflerre-positioningthe 000er on dingpipettein suchaway thatthezonacanbe dissected using the closed microspear. The same on is used to insert the ooplast ~90° lefi the area, which had nedthepolarbody. The zonais closed tightusingthe same tool.

Electmfused cells arewashedandincubatedianTF for40-90minp1iortoICSI.Spermatozoa are immobilized in 10% nylpynolidone (PVP) for ICSI. The procedure is performed in hHTF while the short side ofthe aperture is at approximately 3 o’clock. The ICSI tool is moved throughthe artificial gap htordertoavoidexh'usion ofooplasmupoahidentafion ofthezona duringstandardICSI.Meflndsofimdn'ofertilinationarewellhtownmflieartflouplesue generally first evaluated to diagnose their particular infertility m(s). These may range from unexplained infertility ofboth partners to severe problem ofthe female (e.g., endomtriosis resulting in nonth oviducts with irregular menstrual cycles or polycystic ovarian disease) or the male (e.g., low sperm ommt with morphological abnormalities. or an inability to ate ly as with spinal cord lesions, retrograde ejaculation, or ed omy). The results ofthese evaluations alsodeterminethespecificproeedm'etobeperformed hcouple.

Procedures ofienbeginwiththeadministrationofadmgto down-regulateflie hypothalamiolpituitary system (GnRH agonist). This process decreases serum concentrations of the gonadotropins, and ping ovarian follicles degenerate, thereby providing a set ofnew follicles at earlier stages of development. This permits more precise control ofthe maturation of these new follicles by administration of exogenous gonadotropins in the absence of influences bythe hypothalamicpituitaryaxis.'l‘heprogress ofmaturationandthenmnberofgrowing follicles (usually four-to ten stimulated per ovary) are monitored by daily observations using uloasound and serum estradiol inations. When the follicles attain preovulatol-y size (13.21 mm) and estradiol concentrations continue to rise linearly, the ovulatory response is initiated by exogenous administration ofhuman chorionic gonadotropins 0106).

Following the transplantation prooedm-e, individual oocytes can be evaluated morphologically and transferred to a petri dish containing culture media and heat-inactivated serum. Amensampleisprovidedbythemalepartnerandpmcessedusinga”swimup“ procedure, whereby the most active. motile sperm will be obtained for insemination. Ifthe female's oviducts are present, a procedure called GlFI' (gamete inlrafallopian transfer) can be performed at this time. By this approach, oocyte-cmnulus complexes starotmded by sperm are placed directly into the oviducts by scopy. This procedure best simulates the normal sequences of events and permits fertilization to occur within the oviducts. Not surprisingly, GIFT has the highest success rate with 22% ofthe 3,750 patients undergoing ova val in 1990 having a live delivery. An alternative procedure ZlFT (zygote allopian transfer) permits the selection ofin vitro fertilized zygotes to be erred to oviducts the day following rieval. Exhazygotes canbecryoprescrvcd at'thistimeforfirtm-etranst‘erorfordonation to couples without female gametes. Most ts having more serious infertility problems, however, will require an onal one to two days incubation in culture so that preimplantation ernbryosinthe earlycleavage states canbe selectedfortransfertotheuterus oroviduct. This [VF-UT (in vitro fertilization uterine transfer) procedure entails the transcervical transfer of several 2-6 cell (day 2) or 8—16 (day 3) weimplantation embryos to the ftmdus ofthe uterus (4-5 preimplamation embryos provides optimal success).

Medium for in Vitro fertilization are also described in U.S. Pat. Nos, 6,610,543 6,585,982, 166, 6,352,997, 6,281,013, 6,196,965, 086, 6,110,741, 6,040,340, 6,011,015, 6,010,448, 5,961,444, 5,882,928, 5,827,174, 5,760,024, 5,744,366, 5,635,366, 5,691,194, 5,627,066, 5,563,059, 5,541,081, 5,538,948, 5,532,155. 5,512,476, 5,360,389, ,296,375, 5,160,312, 5,147,315, 5,084,004, 4,902,286, 4,865,589, 4,846,785, 4,845,077, 4,832,681, 4,790,814, 4,725,579, 4,701,161, 4,654,025, 4,642,094, 4,589,402, 434, 4,326,505, 4,193,392, 4,062,942, and 3,854,470, the contents ofwhich are specifically incorporated byrefereneefortheir description ofthese procedures.

Alternatively, patients may elect to haw the oocyte comprising exogenous, autologous OSC mitochondria reimplanwd and fertilized in viva using -inc Insemination (lUl). is swell lmownprocessthatinwlvespreparhrgmd deliveringahighlyconcerrtramdamountof activemotile spermdirectlytln'oughthecervixintotlreuterus.Tllereareseveral ques available for preparing the sperm for IUI. First, sperm is separated from seminal fluid. method ofsperm separation is known as ty Gradient tion”. In this technique, motile reseparated fi'orndeadspermandothercells throughtheuse ofviscous solution. Alter preparation, the oncentrateisplacedtla'oughfliecervixinto theuterusbyusingsthin, flul‘ble catheter and fertilization ofthe reimplanted oocyte follows.

The t invention is additionally described by way ofthe following illustrative, non- lirniting Examples that provide a better understanding ofthe present ion and of its many advantages.

EXAMILES Herein, validated protocols are ed to demonstrate that OSCs can be reliably isolated from tissues ofhealthy young women and propagated in vitro for use in subsequent clinical procedures. The following examples are put forth for rative pin-poses only and are not intended to limit the scope ofwhat the inventors regard as their invention. e 1: BAGS-based Protocol for OSC Isolation The VASA antibody used by Zen et al., Nat Cell Biol 2009 11:631—636 to isolate mouse OSCs by imrnunomagnetic sorting is a rabbit polyclonal t the last 25 amino acids ofthe COOK-terminus ofhuman VASA (DDX4) (ab13840; Abcam, Cambridge, MA). This region shares 96% overall homology with the corresponding region ofmouse VASA (MVH). comparative studies, a goat polyelonal antibody against the first 145 amino acids ofthc NH,- terminus ofhuman VASA (AF2030; R&D Systems, polis, MN) was used, which shares 91% overall homology with the corresponding region ofmouse VASA.

Immunofluorescence analysis ofyoung adult (hounds-old) mouse ovaries using either antibody showed an identical pattern ofVASA expression that was cted, as expected, to oocytes (Figure In). Each antibody was then used for imnumomagnetic surfing ofdispersed young adult mouse ovary tissue (Zou et al., Nat Cell Biol 2009 11:631—636). For each preparation of cells, ovaries lion: 4 mice were pooled and dissociated by g followed by two-step enzymatic digestion involving a 15-minute incubation with 800 U/ml collagenase [type IV; prepared in Hank’s ed salt solution minus calcium and magnesium (11888)] followed by a 10-minute incubation with 0.05% trypsm-EDTA. Digestions were carried out in the presence of l uglml DNascpI (Sigma—Aldrich, St. Louis, M0) to minimize stickiness within the cell preparations, and trypsin was neutralized by addition of 10% fetal bovine serum (FBS; Hyclone. ThermoFisher Scientific, Inc.,Wa1tham, MA). Ovarian dispersates were filtered through a 70—um nylon mesh and blocked in a solution composed of 1% fatty-acid free bovine serum albumin (BSA; Sigma-Aldrich. St. Louis, MD) with either 1% normal goat serum (EMD Millipore, Billerica, MA; for uent reactions using ab13840 t VASA-COOI-l) or 1% normal donkey serum (Sigma-Aldrich, St. Louis, M0; for subsequent reactions using AF2030 against VASA-Nfiz) in I-IBSS for 20 minutes on ice. Cells were then reacted for 20 minutes on ice with a 1:10 dilution ofVASA antibody that recognizes the COOH terminus (ab13840) or NH; terminus (AFZOSO). Afietwards, cells were washed 2 times in I-IBSS and ted for minutes on ice with a 1:10 dilution of either goat anti-rabbit IgG-conjugated microbeads (Miltenyi, Glorlbach, Germany; ab13840 detection) or biotin-configured donkey oat IgG (Santa Cruz hnology, Santa Cruz, CA; AF2030 detection) ed by incubation with stmptavidin—conjugated microbeads nyi; ch, Germany). Alter one additional wash in HBSS, the cell ations were loaded onto MACS columns and separated according to manufacttna’s specifications (Miltenyi, Gladbach, Germany). For experiments to visualize potential antibody-bead interaction with individual oocytes, adult female mice were supcrovulated by ion ofpregnant mare serum gonadotropin MG, 10 IU; Sigma-Aldrich, St. Louis, MO) followed by human chorionic gonadotropin (hCG, 10 IU; Sigma-Aldrich, Louis, MO) 46-48 hours later. Oocytas were collected from oviducts 15—16 hours afler hCG injection, denuded of cumulus cells using hyalm'onidase (Irvine Scientific, Santa Ana, CA) and washed with human tubal fluid (HI'F; Irvine ific, Santa Ana, CA) supplemented with BSA. Dispersed ovarian cells or isolated oocytes were blocbd and incubated with primary antibodies against VASA as described above. Afier washing in HBSS, cells were d with s—appropriate secondary antibodies conjugated to 2.5-um Dynabeada (Invitrogen, Life Technologies Corp., Carlsbad, CA). Suspensions were placed into 1.5 m] Eppendorftubes for separation using a Dyna] MPC°-S Magnetic Particle Concentrator (Dyna! Life Technologies Corp., ad, CA). ' No cells were obtained in the bead fiaction when the VASA-Nflz antibody was used; however, 5-8 tun cells bound to the magnetic beads were observed when the VASA—OOOH antibodywasusedwigure 1h). Analysis ofthese cellsrevealeda germline pression pattern consistent with that reported for OSCs isolated previously by Zen et al., Nat Cell Biol 2009 11:631—636 using immunomagnefic surfing (Figure 2). Although isolated cosy-tag assessad in parallel using the OOH antibody were always detected in the ummoreactive wash fi'action (Figure lb), additional marker analysis ofthe VASA-positive cell fraction obtained by immtmcmagnetic sorting revealed l oocyte-specific mRNAs including Nabax, ZcSand Gdfl(Figtne2).'I'hesefindingsindicate flmtwhilcoocytesdonot exhibitcell surface expression ofVASA when analyzed as individual entities (Figure 1b), oocytes are noneflieless contaminating cell type following innnunomagaetie sorting ofOSCs fiom dispersed ovary tissue. This outcome most likely s either a non-specific physical carry-over of cocytes during the bead centrifugation steps or reactivity of cytOplasmic VASA in plasma membrane- compromised (damaged) oocytes with the COOH antibody. Either case would be alleviated by use ofFACS.

The reactivity ofeach antibody with dispersed mouse n cells was next assessed by FACS. For each experiment, ovarian tissue (mouse: 4 ovaries pooled; human: 10 X10 X 1 mm thick, cortex only) was iated, blocked and reacted with primary dy (abl3840 for VASA—COOH or AF2030 A—NHz) as described above. After washing with HESS, cells were incubated with a 1:500 dilution ofgoat anti-rabbit IgG conjugated to Alexa Fluor 488 (Invitrogen, Life Technologies Corp., Carlsbad, CA; ab13840 Motion) or donkey anti-goat IgG conjugated to Alexa Fluor 488 (Invitrogen, Life Technologies Corp., Carlsbad, CA; AF2030 detection) for 20 minutes on ice, and washed with HBSS. Labeled cells were then filtered again (35-pin pore diameter) and sorted by FACS using a FACSAria It cytometer (BD Biosoiences, Bectun Dickinson and y, Franklin Lakes, NJ; Harvard Stem Cell Institute), gamd against negative (unstained andno primary antibody) controls. Propidium iodide was added to the cell suspension just prior to sorting for dead cell exclusion. Freshly-isolated VASA-positive viable cells were collected for gene sion profiling, assessment atoms formation capacity or in vitro culture. For some experiments, cells were fixed in 2% neutral-buffered paraformaldehyde (PFA) and permeabilized with 0.1% Triton-X100 prior to reaction with primary antibody against the NH; terminus ofVASA (AF2030) and detection by FACS afici- reaction with donkey anti-goat IgG conjugated to Alexa Fluor 488. For re-sort experiments, viable cells were reacted with VASA-COOH antibody (ab13840) and sorted by FACS after reaction with a goat abbit IgG conjugated to allophcocyanin (APC) (Jackson Immrmoreseareh Laboratories, Inc., West Grove PA). Resultant AFC-positive OOK positive) viable cells were then either left intact or fixed and permeabilized prior to incubation with VASA-NH; antibody ), followed by incubation with donkey anti-goat IgG conjugated to Alexa Fluor- 488 and FACS analysts.

In agreement with the magnetic bead surfing results, viable VASA-positive cells were obtained only when the COOH antibody was used (Figmre 1c). However, ifthe n cells were permeabiliaed prior to FACS, a VASA-positive cell tion was obtained using the NH, antibody (Figure 1c). Fmthermore. ifthe viable VASA-positive cells isolated by FACS using the COOH antibody were permeabilized and re-sorted, the same cell population was recognized by the VASA—NH; antibody (Figure 1d). As a final means to confirm validity ofthis OSC method, fi'actions ofcells ateachstep oftheprotocolwere assessedbygeneexpressimanalyais using a cornbination ofmarkers for germ cells (BlimpI/Prdm], Stella/Dppas, Fragllislb‘imd, Terr, Vasa, Dazl) and oocytes (Nabox, Zp3, G199). To obtain cells for FACS, n tissue was minced and enzymatically digested using collagcnase and trypsin. passed through a' 704m filter to remove large tissue , and then passed through a 35—pin filter to obtain a final fiaction s. Every traction ofcells through each step ofthe protocol, with the exception ofthe VASA—positive viable cell fraction obtained by FACS, expressed all gerrnline and oocyte markers (Figure Ii). While the FACS-sorted VASA-posifive cell fraction expressed all a mariners. no oocyte mariners were detected (Figure 1i). Thus, unlike the oocyte contamination observed when 080s are isolated by immunornagnetic surfing using the VASA—GOOH antibody (see Figure 2), use _sarne antibody with FACS provides a superior strategy to obtain adult ovary-derived OSC ns free of oocytes.

Example 2: Isolation of OSCs From Human Ovaries Withvaitteninformedoonsent, ovariesweresln-gicallyrelnoved fi'om6 femalepatierlts between 22—33 (28.5 :1: 4.0) years ofage with Gender Identity Disarder for sex reassignment at Saitama Medical Center. The outer cortical layer was carefillly removed, vitrified and cryoprcserved a et al., Reprod. Biamed. 2009 Online 18:568-577; Figln-e 12). Briefly, 1 min-thick cortical fiagments were cut into rn1| (10 X10 mm) pieces, incubamd in equilibration solution ning 7.5% ethylene glycol (EG) and 7.5% dimethylsulfoxide (DMSO) at 26° C for 25 minutes, and then incubmd in a vitrification solution containing 20% EG, 20% DMSO and 0.5 M sucrose at 26° C for 15 minutes priorto submersion into liquid nitrogen. For mental analysis, cryopreserved ovarian tissue was thawed using the smeThawmgIfitmitamtoBiophamFujiCityfihimolm, Japan)andprocessed immediately for histology, xenografiing or OSC isolation. Using flue COOH antibody, viable VASA-positive cells between 5—8 pm in diameter were also consistently isolated by FACS fi'om human ovarian cortical tissue biopsies of all patients n 22—33 years ofage, with a percent yield (1.7% :l: 0.6% VASA-positive versus total viable cells ; mean i SEM, n = 6) that was comparable to the yield ofOSCs from young adult mouse s processed in parallel (1.5% :l: 0.2% VASA-positive versus total viable cells sorted; mean :I: SEM, n = 15). This t yield is the incidence ofthese cells in the final pool ofviable single colll sorted by sacs, which representsai'raclion ofthemtalnumberofcellspreserltinovariespfiortoprocessing, To eatinmte the incidence ofOSCs per ovary, the c DNA contentper ovary of 1.5—2 month- old mice was determined (1,774.44 :1: 426.15 pg; mean :1: SEM, n = 10) and divided into genomic DNA content per fraction ofviable cells sorted per ovary (16.41 i: 4.01 pg; mean :1: SEM, n = 10). Assuming genomic DNA content per cell is equivalent, how much ofthe total ovariancell pool isrepresentedbyflletotalviable sortedcell fraction obtainedafterprocessing was determined. Using this correction , the incidmee ofOSCs per ovary was estimated to be 0.014% :I: 0.002% [0.00926 X (1.5% :t 02%)]. With respect to OSC yield, this number varied across replicates but between 250 to slightly over 1,000 viable VASA-posifive cells per adult ovary were consistently obtained alter FACS of dispersates lly prepared from a pool of4 ovaries.

' Analysis offi'eahly-isolated VASA-positive cells from both mouse and human ovaries (Figure 3a, 3b) revealed a similar size and logy (Figure 3c, 3d), and a matched gene sion profile rich in markers for early germ cells (Salton et al., Nature 2002 418:293—300; Ohinata et al., Nature 2005 436:207—213; Dolci et al., Cell Sci. 2002 115:1643—1649) (Blbnpl, Stella, Fragilir and Terr; Figure 3e). These results agree with the morphology and gene expression profile ofmouse 0505 reported in the scientific literature (Zou et al., Nat CellBiol 2009 —636. Pacchiarotti et al., Dgfi'erenriation 2010 79:159—170).

To r define characteristic features ofVASA-positivc cells obtained fi'om adult ovaries, mouse OSCs were tested using an in viva teratoma ion assay. This was important since a recent study has reported the isolation of 0ct3/4-positive stem cells fi‘om adult mouse ovaries that possess the teratoma-forming capacity of embryonic stem cells (E805) and induced pluripotent stem cells (iPSCs) (Gm-lg et al., Fertil. Steril. 2010 93:2 01). s were collected fine: a total of 100 young adult female mice, dissociamd and subjected to FACS for isolation ofVASA—COOH positive viable cells, as described above. Freshly ed mouse OSCs were injected subcutaneously near the rear haunch ofNOD/SCH) female mice (1x105 cells injected per mouse). As a control, mouse embryonic stem cells (mESC v6.5) were injected into age-matched female mice in parallel (lxlos cells ed perrecipient mouse). Mice were monitored weekly foruptononths fortrmrorformaticn.

As molested, 100% offltemiceh‘ansplantedwifllmousc ESCsusedas ivecontrol developed terattomas within 3 weeks; however, no teratomas were observed in mice transplanmd in parallel with ositive cells isolated from adult mouse ovaries, even at 24 weeks post- transplant (Figunes 3f—lr). Thus, while OSCs elrpress numerous stem cell and primitive germ cell markers (Zou et al., Nat Cell Biol 2009 11:631~636, Pacehiarotti et a1., Diflierentiation 2010 79:159—170; see also Figure if and Figure 3e), these cells are clearly distinct fiom other types of pluripotent stem cells described to date.

Example 3: Convention ofOocytes from FACS-parified mouse osc.

The ability ofFACS-purified mouse OSCs, engineered to express GFP through retroviral transduction (alter their establishment as actively-dividing germ cell-only cultln'es in vine) to generate s ing nonsplsntation into ovaries ofadult female mice was assessed. To ensure the outcomes obtained were reflective ofstable integration ofthe transplanted cells into the s and also were not camd by pre-tnnsplantafion induced damage to the gonads, 1x10‘ GFP-expressing mouse OSCs were injected into ovaries ofnon- chemotherapy conditioned wild-type recipients at 2 months of age and animals were maintained for 5—6 months prior to analysis. Between 7—8 months ofage, transplanted animals were induced to ovulate with exogenous gonadotropins (a single intrapuitoneal injection of PMSG (10 IU) ed by hCG (10 IU) 46—48 hours later), after which their ovaries and any oocytes released into the oviducts were collected. Ovulated culnulus-oocyte complexes wetransferred into HTF supplemented with 0.4% BSA, and assessed by direct fluorescence microscopy for GFP expression. ping follicles containing GFP-positive oocytes Were readily detectable, along with follicles containing GFP-negative oocytes, in s of females that ed GFP- expressing mouse OSCs initially purified by FACS (Figure 4a).

After oviductal flushing, complexes corrteining ed cumulus cells nding centrally-located s both lacking and expressing GFP we observed. Mixing ofthese complexes with sperm from ype males resulted in fertilizatim and development of preimplantstion embryos. For in vitro fertilization (IVF), the cauda epididymides and vas deferens were removed from adult wild-type 6 male mice and placed into HTF medium supplemented with BSA. Sperm were obtained by gently squeezing the tissue with twesners. capaeitnted for 1 hour at 37' c, and then mixed with commas-00cm complexo (1—2 x 10‘ sperm/ml in H'I'F medium supplemented with BSA) for 4—5 hours. Inseminated oocytes were then washed ofsperm and transferred to flesh medium. At 4—5 hauls post-insemination, cocytes ‘10 (fertilized and unfertilized) were erred to 50 u] drops ofKSOM-AA medium (Irvine Scientific, Santa Ana, CA), and the drops were covered with mineral oil to support flu'ther preimplantafion nic development. Light and fluorescence microscopic examination perde every 24hours foratotal of l44hours tomonitorembryo developmentto the hatching blastoeyst stage (Selesniemi et al., Proc. Natl. Acad. Sci. USA 2011 108:12319—12324).

Ovarian tissue harvested at the time of ovulated oocyte collection fi'om the oviducts was fixed and processed for immunohistccheucal detection ofGFP expression using a mouse monoclonal antibody against GFP (sc9996; Santa Cruz Biotechnology, Santa Cruz, CA) along with the MOM” lrit (Vector Laboratories, Bmlingame, CA), as detailed usly (Lee et al., J. an Oncol. 2007 25:3198—3204). Ovaries fi'om non~transplanted wild-type female mice and final TgOG2 transgenic female mice served as negative and ve ls, respectively, for GFP detection.

Preimplantation embryos derived fi'cm fertilized GFP-positive eggs retained GFP expression through the hatching blastocyst stage (Figure 412—11). From the 5 adult ype female mice transplanted with GFP-expressing OSCs 5-6 months earlier, a total of 31 annulus- oocyte complexes were retrieved from the oviducts, 23 ofwhich succeslfiilly fertilized to produce embryos. The presence lus cells aromd each 000th made it impossible in accmately ine the numbers ofGFP-negative versus GFP-apositive oocytes ovulated.

However, evaluation ofthe 23 embryos produced following in vitra fertilization (IVF) revealed that 8 were GFP-positive, with all 5 mice tested ing at least one eg at ovulation that fertilized to produce a GFP-positive embryo. These findings indicate that OSCs isolated purified by VASA—COOH antibody-based FACS, like their usly reported counterparts isolated by innmmomagnetic surfing (Zou et al., Nat Cell Biol 2009 11:631—636), generate fimcfional oocytes in viva. However, our data also show that chemotherapy conditioning prior to transplantation is not, as previously reported (2011 et al., Nat Cell Biol 2009 11:631-636), required for OSCs to engrait and generate functional oocytes in adult ovary tissue.

Example 4:1» vino Characterization of Candidate Human OSCs Using parameters described previously for in vitra propagation ofmouse OSCs (Zou et al., Nat Cell Biol 2009 11:631—636), adult mouse and human ovary-derived VASA-positive cells were placed into defined cultures with mitotically—inacfive mouse embryonic fibroblasts (MEFs) as feeders. Briefly, cells were cultured in MEMu. (lnvitrogen, Life Technologies Corp, Carlsbad, CA) supplemented with 10% FBS ne, ThermoFisher Scientific, Inc., Waliham, MA), 1 mM sodium pyruvate, 1 mM sential amino acids, IX-concentrated penicillin- sh'eptomycin-glulamme (Inviu'ogen, Life Technologies Corp., Carlsbad, CA), 0.1 mM B- ma-eaptoeflianol (Sigma, St. Louis, MO), centrated N-2 supplement (R&D Systems, Minneapolis, MN), ia inhibitory factor (NF; 103 mils/ml; EMD Millipore, lnc., Billerica, MA), 10 nglm] recombinant human epidermal growth fictor ; lnvitrogen, Life Technologies Corp., Carlsbad, CA), 1 ng/ml basic fibroblast growth factor (bFGF; gen, Life Technologies Corp., Carlsbad, CA), and 40 nyml glial cell-derived neln'otropic factor (GDNF; R&D Systems, Minamolis, MN). Culun'es were refreshed by the addition of40-80 ofnew medium every other day, and cells were re-plated on flash MBFS every two weeks. To assess proliferation, MEF-fi'ee OSC culunes were treated with 10 LLM BrdU (Sigma-Aldrich, Louis, MO) for48 homspliortofixafioninZ'Ml PFAfordual inmumofluorescence-based detection ofBrdU incorporation (mitotically—acfive cells) and VASA sion (germ cells), as desoribed(Zou et at; Not CeIIBi012009 11:631—636). No signal was detected ary antibodies were omitted or replaced with an equivalent dilution ofnormal rabbit serum (not shown). ' Freshly-isolated OSCs could be ished as clonal lines, and the colony formation eficiency for SCs not seeded nto MBFsranged fi'om0.18% to 0.40%. Accurate assessment ofcolony fimmfim efiiciency could not be performed using MEFs as initial s, the latter ofwhich greatly facilitates esmblislnnmt ofmouse and human 0803 in via-a. Alter —12 weeks (mass) or 4—8 weeks (human) in culture, actively-dividing germ cell colonies became readily apparent (Figure 5). Once ished and proliferating, the cells could be re- established as germ cell-only es in the e ofWe without loss of proliferafive potential. Dual analysis ofVASA expression and bromodnoxyuridine (BrdU) incorporation in MEF-free cultures revealed large numbers of double-positive cells (Figure Ga—d), confirming that adult mouse and human ovary-derived VASA—positive cells were actively dividing. At this stage, mouse cells required passage at confluence every 4-5 days with cllltla-es split l:6—l:8 (estimated doubling time of 14 hours; Figure 6c). The rate ofmouse OSC proliferation approximately 2—3 fold higher than that ofhuman germ cells maintained in parallel, the latter of which required e at confluence every 7 days with 'es split 1:3—1 :4.

Cell surface expression ofVASA remained detectable on the sin-face ofmore than 95% ofthe cells utter months ofpropagation (Figure 6f). The remaining cells not detected by FACS using the VASA- COOH dy were large (35—50 pm in diameter) spherical cells spontaneously produced by mouse and human OSCs during culture, which exhibited asmic expression ofVASA and are described in detail in Example 5.

Gene expression analysis of the cultured cells confirmed maintenance of early germline markers (Figure 6g). l oocyte-specific markers were also detected in these cultures.

Levels ofmRNA were assessed by RT-PCR using a SuperScript® VILOTM cDNA Synthesis Kit (Invitrogen, Life logies Corp., Carlsbad, CA) and Platinum Taq rase (Invitrogen, Life Technologies Corp., Carlsbad, CA). All products were sequenced to confirm identity. Sequences of forward and reverse primers used, along with GenBank accession s ofthe corresponding genes, are provided in Table 1 (mouse) and Table 2 (human).

Table 1. PCR rimers used to anal ze ene ex ression in mouse cell and tissue sam les.

Primer sequences(5’ to 3’; F, forward; R, SEQ ID Size Gene Accession No. e NOS . .) ___—— ___-.- ___—— ___“151 ___-- ___—_- ___-.— F: GGAAACCAGCAGCAAGTGAT _—R:TGGAGTCCTCATCCTCTGG _- Dazl F: GTGTGTCGAAGGGCTATGGAT 328 -—R:ACAGGCAGCTGATATCCAGTG _— F: CCTCCCCACTTTCCCATAAT _—R:AATGGGTGGGGAAGAAAAAC _— F: AGCAGAGAGCTTGGTCGGG _—R:TCCGGTGAGCTGTCGCTGTC _— F: CTCACGCTTCCACAACAAGA _E- ___-II— ___—— ___—— ___—— _—R:TGATGGTGAAGCGCTGATAG _— __R:GGGTGGAAAGTAGTGCGGTA _— F: CCGAGCTGTGCAATTCCCAGA Table 2. PCR rimers used to anal ze ene ex ression in human cell and tissue sam les. ersequences(5’ to 3’; F, forward; R, SEQ ID Size Gene Accession No. reverse) NOS (bp) _——_- _———_ _—__- _——_- _——_- ——R:TAGGATTCATCGTGGTTGTGGGCT _- F: TAAACGCCGAGAGATTGCCCAGA _—R:AGTCTGGTCAGAAGTCAGCAGCA _- NM 001001933 _——_- _——_- _——_- _——_- l--_m_ _—R:CTCCTTAATGTCACGCACGAT _- To extend the mRNA analyses ofBlimp] Stella and Fragilz's, immunofluorescence analysis of these three classic primitive germline markers was performed (Saitou et al., Nature 2002 418:293—300; Ohinata et al., Nature 2005 436:207—213). For analysis of cultured OSCs, cells were washed with lX-concentrated ate-buffered saline (PBS), fixed in 2% PFA for 45 minutes at 20° C, washed 3 times with PBS-T (PBS containing 0.01% Triton-X100) and incubated for 1 hour at 20° C in blocking buffer (PBS containing 2% normal goat serum and 2% BSA). The cells were then incubated for 1 hour at 20° C with a 1:100 dilution of one of the following primary antibodies: a biotinylated mouse onal against BLIMPl (ab81961, Abcam, Cambridge, MA), a rabbit polyclonal against STELLA (abl9878; Abcam, Cambridge, MA) or a rabbit polyclonal against FRAGILIS (mouse: ab15592, human: ab74699; Abcam, Cambridge, MA). Cells were washed and incubated for 30 s at 20° C with a 1:500 dilution of steptavidin-conjugated Alexa Fluor 488 (Invitrogen, Life Teohn ologies Corp., ad, CA; BLIMPl detection) or goat anti-rabbit IgG conjugated to Alexa Fluor 488 (STELLA and FRAGILIS ion) in the presence ofrhodamine-phalloidin (InVitrogen, Life Technologies Corp., Carlsbad, CA). Cells were washed, incubated with 4',6-dimnidmophenylmdole dihydrochloride (DAPI; Sigma-Aldrich, St. Louis, MO) and washed 3 additional times before imaging. No signal was detected ifprimary antibody was omitbd orreplaced rmal serum (not .

For assessment of oocytes generated in vitro by mouse and human OSCs, individual 000th8 were collected from culturemam, washed, fixed with2% PFA mammg'om BSA for 45 minutes at 37' C, washed and bloobd for 1 hour at 20' C in PBS containhg 0.5% BSA and either 5% normal goat serum (VASA or LIDCB detection) of 1% normal donloey semm (c—KIT demotion). Aficrblocking, ooeytaes were incubated for 2 hours at 20‘ C with a 1:100 dilution (in PBS with 0.5% BSA) of one ofthe following primary antibodies: :1 goat polyclonal against c-KIT (sc1494. Santa Cruz Biotechnology, 1110., Santa Cruz, CA). a rabbit polyclonal tVASA (ab13840, Abcam, Cambridge, MA) or a rabbit polyclonal againstLHXS (ab41519, Abcam, Cambridge, MA). Cells were then washed and awd with a 1:250 dilution ofgoat abbit IgG conjugated to Alexa Fluor 568 (Invitrogen, Life logies Corp, ad, CA; VASA detection) or Alexa Fluor 488 (LI-1X8 detection), or a 1:250 dilution ofdonkey anti-goat IgG conjugated to Alexa Fluor 488 (c~KIT detection). Cells were , incubated with DAPI and washed 3 additional times before imaging. No signal was detected ifprimary antibody was omitted or replaced with normal serum.

For these latter experiments, detection of oocyte-speeific sion ofVASA, c-KII‘ and, for human ovaries. LHXS in ovarian tissue sections med as a ve control. Mouse and human ovarian tissue was fixed in 4% PFA, parafi'in—embedded and sectioned (6-pm) prior to high ature antigen retrieval using 0.01 M sodium citrate bUfl‘er (pH 6.0). After cooling, sections were washed and blocked for 1 hour at 20' C using TNK buffer (0.1 M Tris-H01, 0.55 MNaCl, 0.1 mM KCL, 0.5% BSA, and 0.1% Triton-X100 in ate-bufi'ered saline) containing either 1% normal goat sennn (VASA-COOH or LHXB detection) or 1% normal donloey set-Inn NASA-NH; or o-KIT detection). Sections were then incubm with a 1:100 dilution ofprimary antibody (in TNK buffer with 1% normal serum) overnight at 4' C, washed in PBS, and incubated for 30 minutes at 20' C with a 1:500 dilution of goat anti-rabbit conjugated to Alexa Fluor 568 (VASA-COOH detection in human ovary), goat anti-rabbit IgG conjugamd to Alexa Fluor 488 (detection ofVASA-COOH in mouse ovary or LHXS) or donkey anti-goat IgG conjugated to Alexa Fluor 488 (c-KIT or VASA-NH: detection). Afier washing with PBS. sections were cover-slipped using Vectashield containing DAPI (Vector Labs). signal was detectedifprimary antibodywas omittedorreplaced withnormfl serum.

All flrreeproteinswereeasilyandmiifonnly detected inmouse (Figure 6h) an (Figure 6i) OSCs maintained in ultra. Notably, detection ofFRAGIIJS in these cells agrees with areeentstudyreportingthatthisprotein canalsobeusedtoisolate OSCs fiommouse sby nnmunomagnetic bead sorting(Zou et al., Stem Cells Dev. 2011 doi: 9/scd201 1.0091).

Example 5: In with; Oogenlc Capacity of Candidate Human OSCs‘ Consistent with results fi'om others (Pacehiarotli et al., Diferenfiatian 2010 —170), mouse OSCs cultured in vitro neously generated large (35—50 gun in diameter) spherical cells that by morphology (Figure 7a) and gene expression analysis (Figure 71:, c) resembled oocytes. Peak levels ofin vilra oogenesis fi'ommouse DSCs were observed within 24-48 hours alter each passage (Figure 7d), followed by a ssive decline to nearly non-detectable levels each time OSCs regained confluence. Parallel analysis -positive cells isolated from adulthuman ovaries and maintained in vitro revealed that these cells, like mouse OSCs, also spontaneously generated oocytes as deduced from both morphological e 71) midgeneeamressionwigme'lc, g)analyscs.‘l‘helcinefies ofinvltra oogenesis i'romhuman OSCs difi'ered slightly frommouse osc5 in that peaklevels ofoocyua formation ’ were observed at 72 hours alter ssage (Figure 7e). In addition to detection of many widelyaccepted oocyte markers (Vasa, o-Kit, Nobox, th8, GM. Z01, sz, Z03; ri et 31.,Mech. Dev. 2002 111:137—141;Rajkcvic et al., e 2004 305:1157—1159; Pangas et al., Proc. Natl.

Acad. Sci. USA 2006 103:8090-8095; Elvin et al., Mol. Endocrinol. 1999 13:1035-1048; Zheng et al., Semin. Reprod. Med. 2007 25243—251), mouse and human USO-derived oocytes also expressed the diplotene ooeyte stage-specific markerM92 (Figure 7c). MSY2 is a mammalian gue ofXenapm FRGYZ, a germ cell-specific nucleic acid-binding Y—box protein that is essential for meiotic progression and gametogenesis in both sexes (Gu et al., Biol.

Reprod. 1998 59:1266—1274; Yang etaL, Prac. Natl. Acad. Sci. USA 2005 102:5755-5760). Through empirical testing ofcommercially-available antibodies using adulthuman ovarian cortical tissue as a ve control, four such antibodies against oocyte s Were identified that ' specifically reacted with irmmture oocyms present in adult human ovnries (VASA, c-KIT, MSY2,LHX8; Figure 8); all fouroftheseproteinswerealsodetectedinoocytes generatedby human OSCs in vitro e 7g). passage identified cells with punctate nuclear localization of the meiosis-specific DNA recombinase, DMCl, and the meiotic recombination protein, synaptonemsl complex protein 3 (SYCPS) (Figure 7h). Both proteins are specific to gem: cells and are necessary for meiotic recombination (Page et al., Annu. Rev. Cell Dev. Biol. 2004 20:525-558; Yuan et al., Science 2002 296:1115—1118; Kagawa et a1., FEBSJ. 2010 277:590—598).

Chromosomal DNA t analysis ofhuman OSC cultures 72 hours afier passage was detcmu'ned. Cultured mouse (48 hours after passage) or human (72 hours aflrr passage) OSC: were collected by trypsinization, washed and resuspended in ice-cold PBS, and counted with hemocytometer. Afier fixation in ice-cold 70% ethanol for 1 hour, cells Were washed in ice-cold PBS and incubated with 0.2 rug/ml RNase-A for 1 hour at 37' C. Propidium iodide was then added (10 gig/ml final), and ploidy status was determined using the BD Biosciences FACSAria lI cytometer. As a control somatic cell line, these experiments were ed using human fetal kidney fibroblasts (l-IEK 293, Invitrogen, Life Teclmologies Corp., ad, CA). This analysis ed the ce ofan ted diploid (2n) cell population; however, peaks corresponding to 4:: and In populations ofcells were detected, the latterbeing indicative ofgerm cells that'had reachedhaploid status (West et 31., Stem Cells Dev. 2011 20:1079— 1088) (Figure 7i). In ly- dividing cultures of fetal human kidney fibroblasts analyzed as controls in parallel, only 2n and 4n populations of cells (Figure 9a) were detected. Comparable outcomes We observed ing FACS-based chromosomal analysis ofmouse OSC cultures (Figure 9b).

Example 6: Human OSCs Generate Oocytes in Human Ovarian Cortical Tissue In Vivo To confirm and extend the in w‘tra observations ofputative oogeneais fi‘om candidate human OSCs, in two final experiments VASA—positive cells isolated fi-om adult human ovaries were stably u’ansduced me a car expression vector (GFP-hOSCs) to narrate cell insulting.

ForeefluacldngexpefimenmhumanOSCswereu'ansducedusing areuovirustoobtain cells with stable expression ofGFP OSCs). Briefly, 1 [Lg oprabe-(bjz vectorDNA (Addgene plasmid repository #10668) was transfected as per the manufaehlrer’s protocol (Lipofectamine, Invitrogen, Life Technologies Corp., Carlsbad, CA) into the Platinmn-A retroviral ing cell line (Cell Biolabs, Inc., San Diego, CA). Viral supernatant was collected 48 horn-s afler transfection. Transduction ofhuman OSCs was med using fresh viral supernatant facilitated by the presence ofpolybrene (5 fig/ml; Signs-Aldrich, St. Louis, MO).

Afier 48 hours, the virus was removed and replaced with flash OSC culture medimn. Human OSCs with expression ofGFP were purified or isolated by FACS following an initial 1 week ofexpansion, and the purified or isolated cells were expanded for additional 2 weeks befiore a second round of FACS purification or surfing to obtain GFP-hOSCs for human ovarian tissue re-aggregation or afling experiments.

In the first experiment, approximately 1 X 10’ GFP-hOSCs were then m—aggregataed with dispersed adult human n cortical tissue. Human ovarian cortex was iated and washed as described above, and incubated with 35 ug/ml emaglutannin (PHA; Sigma, St.

Louis, MO) plus 1x105 GFP~hOSCs for 10 minutes at 37' C. The cell mix was pelleted by centrifugation (9,300 xg for 1 minute at 20‘ C) to create the tissue aggregate, which was placed onto a Millicell 0.4 um culture plate insert (END) Millipore, Inc.. Billerica. MA) contained in 6-well 0111th dish with 1 ml ofOSC culture medium. Aggregates Wereincubated at 37' C in 5% Gog-95% air, and live-cell GFP imaging was performed 24, 48 and 72 hours later.

Numerous GFP-positive cells were observed, as expected. throughout the regated tissue (Figure 10a). The aggregates were then placed in culture and assessed 24—72 hours later by direct (live cell) GFP fluorescence. Within 24 hours, l very large (EEO-pm) single cells were also observed in the aggregates, many ofwhich were enclom by smaller GFP-ncgative cells in tightly t structures resembling follicles; these strictures remained detectable through 72 hours (Figure 10b. c). These findings indicated that GFP-expressing human OSCs spontaneously generated oocytes tlnt became enclosed by somatic (pregranulosalgranulosa) cells present in the adult human ovarian dispersates.

Next, GFP-hOSCs were injected into adult human ovarian cortical tissue biopsies, which were then xenografled into NOD/SClD female mice (n= 40 gratis total). Ovarian cortical tissue pieces (2 X 2 X 1 mm) were individually injected with approximately 1.3 X 10’ GFP-hOSCs using a lO-ul NanoFil syringe with a 35-gauge beveled needle (Woo-1d Precision Instmments, Ssrasota,FL).RecipiorrtNOD/SCID femalemioewere anesflietizedandasmall incisionwas madealongthcdorsal flankforsubcutaneous insertionofthehmnanovariantissue, elsentiully as descn'bed (Weissman et al., Biol. Reprod. 1999 60:1462—1467; Matilminen et al., Nature Genet. 2001 28355—360). Xenografis were removed after 7 or 14 days post transplantation, fixed in4% PFA, n-embedded and serially ned ) for immimohistochemical analysis using a mouse monoclonal dy against GFP (sc9996; Santa Cruz Biotechnology.

Santa Cruz, CA) (Lee et al., J. Clin. Oneal. 2007 25:3198—3204). Briefly, high ature antigen retrieval was rformcd using 0.01 M sodium citrate buffer (pl-I 6.0). Afie- g. sectionswere ted for 10 minutes with 3% hydrogen peroxide in methanol to block endogenous peroxidase activity, washed and incubated in streptavidin-biotin pre-block solution as per the manufacun'er’s protocol (Vector Laboratories, Burlingame, CA). Sections were then for l hourat20' Cusing'lNKbufmrcontaining 1%normalgoatserumandincubated ght at 4' C with a 1:100 dilution of GFP antibodyprepared in THE buffer containing 1% normal goat serum. Sections were then washed, incubated with a 1:500 dilution of goat anti- mouse biotinylated secondary dy for 30 minutes at 20' C. washed and reacted with Vectastain ABC reagents (Lab Vision, ThermoFisher Scientific, Inc., m, MA) for 30 and tissue architecture. Negative controls (complete immmohistochemical staining protocol on xenografied tissues that received vehicle injections) were always nm in parallel and did not show a positive signal. To confirm and extend these observations, dual ilnmunofluorescence-based demotion ofGFP and eitherMSY2 (diplomne stage oocyte—specific marlmr) or LHXS (early stage oocyte ription factor) in afied human ovarian tissues was med with DAPI counterstaining, as detailed previously in the description ofilmmmoanalysis.

Grafts were ted 7 or 14 days later for assessment ofGFP mession. All human ovary grafts contained easily discernible primordial and primary les with centrally-located GFP-neptive oocytles. Interdispersed among and often adjacent to these follicles, which were presumably present in the tissue prior to GFP-hOSC injection, were other immature follicles containing GFP-positive oooytes (Figure 10d, 1). Serial section hiatomorphornetric analysis of 3 randomly selected human ovarian tissue biopsies injected with GFP-hOSCs revealed the presence of 15-21 GFP-positive oocytes per graft '7 days after xenografting into mice (Figure 11). As ls, GFP-positive oocytes were never detectedin human ovarian cortical tissue prior to GFP-hOSC injection (Figure 106) or in xenografis that received mock injections le without GFP-hOSCs) prior to transplantation into NOD/SCID mice (Figure 10g). Dual fluorescence—based detection ofGFP along with either the diplotene stage oocyte- specific marker MSYZ (Go et al., Biol. Reprod. 1998 59:1266-1274; Yang et al., Prac. Natl.

Acad. Sci. USA 2005 102:5755—5760) or the oocyte-specific transcription factor LHX8 (Fungus et al., Pros. Natl Acad. Sci. USA 2006 103:8090—8095) identified many dual-positive cells distribumd throughout xenogrsfis ed with GFP-hOSCs (Figure 10h). As ed, no GFP- ve oocytes were ed in ovarian tissue prior to SC injection or in xenogmfts that did not receive GFP-hOSC injections (not shown; see Figure lOe, g); however, these oocytes were consistently positive for LHXS and MSYZ (Figure 10h; Figure 8).

Example 7: Use ofOSCs In gons Germltns Mitochondrial Energy Transfer (“AUGMENT”) Figure 13 depicts an overview ofthe use ofOSCs as an autologous source of female germ cells for derivation ofoogenio cytoplasm or mitochondrial freedom: that can then be transferredintoanoocyte obtainedfi‘omthe same subjectpriorto ordlning in vitro fertilization (IVF). The resultant boost in mitochondrial DNA copy number and ATP-generating capacity in the eg after AUGMENT ensm'es that the oocyte has ample teem ofATP for energy-driven events required for successful fertilization and embryonic development. The additional nfimhmdfiamovidcdmthemcytebyAUGMENdeedwdfimthenamalmecmsmcen used by the body to produce oocytcs. Furthermore, the additional mitochondria will not produce adverse effects in the oocyte, based on data showing that healthy embryogenesis proceeds even when the minimal threshold number ofmitochondria needed for embryo development is exceeded by nearly four-fold (see Wei et 31., Biology ofReproduction 2010 83:52-62, Figure 6).

The beneficial efi‘ects ofheterologous ooplasmic transfer reported earlierby Cohen et 111., Mol Hum Reprod 1998 4:269—80, a procedure which is not suitable for human use because it results in germline genetic manipulation and mitochondrial heteroplasmy in embryoslofl’spring, indicate that oocytes are benefited by additional mitochondria.

An exemplary al protocol for AUGMENT is as folloWs. Prior to the start of standard NF. the subject will undergo a laparoscopy during mstl'ual cycle days 1-7 to t up to three pieces (approximately 31:31:! mm each) ofovarian epithelium (ovarian cortical biopsy) fi'om one ovary. During this procedure, 2-3 ons will be made within the abdomen anda device willbeinsertcdtoremovethe tissucfiomanovaryusingsterile procedures. tissue collected will be placed in sterile on and transported on ice to the GTP compliant laboratory where it will be cryopreserved until the time ofAUGMENTIICSI. The tissue will remainfi'ozenrmtilthetimeofenzymatic dissociation. 'I‘hiswillserveasthesomce of autologous OSCs fi'om which mitochondria will be pmified or isolated.

Nat, OSCs will be isolated and ondria will be harvested item the 0808. After thawingthe ovariancorficflbiopsiedfissueflretissuewillbemincedandplacedin solution, containing recombinant collagenase and recombinantDNasel and homogenized to a single cell suspension. 'Ihesuspensionwillbepassedtln'oughacellsuainerto prepareasolufionofsingle cells. The single cell suspension will be incubated with an anti-VASA antibody. d cells will then be isoWd by fluorescence-activated cell sorting (FACS). Standard slow cooling cryopreservafionprocedm'es forfi'eezingaliquots ofOSCs willbeused.

Subjects will o ardIVFprotocol includingbaselineevaluation, GnRH antagonist egulation and gonadotropin stimulation. Oocyte retrieval will take place within 34-38 hours afiel- hCG administration and oocytes will be assessed for quality and marmation state. Mature oocytes will be inseminated by 1081.

Onthe dayofoocyteretrieval, thefrozenOSC vial forthat subjectwillbethawed using standard methods. 0805 will be promed to yield a mitochondrial pellet (Frezza et al. Nature Protocols 2007 2:287-295 or Perez et al., Cell Death andDifl'erentr'otion 2007 3:524-33.

Epub 2006 Oct 13) or as bed below in Example 9, where a FACS-based method is employed to isolate the total mitochondrial population in a tissue and optionally, tin-flier isolate the actively respiring ondrial population or quantitate the ratio of active to total mitochondria in a . Evaluation and ty ofthe mitochondrial preparation will be assesm and recorded.

Exemplary assays ofmitochondrial n are described in Example 8. The mitochondrial pellet will be re-suspended in media to a standardmd concentration ofmitochondrial activity which improves oocyte quality. This media containing the mitochondria will be ted into a microiniection needle that contains the spermatozoan to be delivered. Both the mitochondria and spermatozoan will be delivered together into the cocyte by ICSI. Alternatively, the mitochondria or preparation thereofwill be from prior to use.

Following fertilization and embryo culture, typically a maximum ofthree, grade 1 or grade 2 (SART grading system (50)) embryos may be tansferred under ultrasound guidance alter 3 or 5 days of culuuing based an the assessment of embryo development. He pregnancy is confirmed via beta hCG testing, then the subject will have subsequent observations at approximately 6 and lit-weeks gestational age.

Example 8: Assessment of Mitochondrial Parameters in Human OSCs versus Human Ovarian Stromal Cells. ' Mitochondrial staining was conducted in cultured human ovarian somatic cells and cultured human OSCs obtained fi'om the same patient. Cells were incubated with the non~ oxidation dependent MitoTracker Green FM (Invitrogen, Life Technologies Corp., ad, CA; M7514) mitochondrial ng probe, which tes mitochondrial mass, at 37' C for 45 minutes and washed twice with fresh culture medium prior to live cell fluorescent imaging.

Both cell types were processed in parallel. In Figure 14, distribution patterns indicate perhruelear localization in the human OSCs, consistent with other hurmn stem cell types.

Accumulation ofa connnon deletion mutation (deletion ofnucleotides 3447 of themitochondrion genome) occursinrntDNA ofcells as an organist-rages, PCRprimers Were designed to span this deletion. If the deletion mutation is absent. indicating the mtDNA genome is intact, the PCR amplieon will be 5080 bp. Iftiie deletion is present, a 103 bp fragment will amplified. In instances rogeneity among the mitochondria Within individual cells or the cell population, both products do not y. This occurs because because the deletion (as md by the anal] band) amplifies much more eficiently than the large 5-kb product. The small product s the exponential and plateau phases more rapidly, thereby utilizing the available reagents in the PCR mix and g little or none for the less efficient S-kb product amplification. The PCR. analysis shown in Figure 15 ms that the human 0303 do not harbor an accumulation ofthe mutation, whereas patient matched ovarian somatic cells do.

To confirm that the mitochondrial population within the somatic cells is geneous with respect to the mutation (some mitochondria will harbor the deletion and some will not), a second set ofPCR s targeting a sequence specifically within the deleted region was used to assess mitochondrial integrity in ovarian c cells. The amplification of a 191 bp product indicates that this region is intact within at least some ofthe mitochondria in time cells. and that the overall population ofsomatic cell mitochondria is heterogeneous with t to the deletion on, whereas the human 030s are essentially fine ofthe mutation.

Primer sequences (5’ to 3’) for mitochondrial DNA analysis include on l for 5080 bp t) or 103 bp (deletion mutant) having the following sequences: TTACACTA'I'I‘CCTCATCACCCAAC (SEQ ID NO: 64) (forward) and TGTGAGGAAAGGTATTCCTGCT (SEQ ID NO: 65) (reverse) and Amplicon 2 for 191 bp (internal, deleted ce) having the ing sequences: CCTACCCCTCACAATCATGG (SEQ ID NO: 66) (forward) and ATCGGGTGATGATAGCCAAG (SEQ ID NO: 67) (reverse).

Figure 16 depicts the results of an ATP assay (ATP Bioluminescence Assay Kit HS H, Roche Applied Science, Mannheim, Germany). The left panel shows the standard curve following dilution of ATP (molar ratio vs. chemiluminescence). As shown, the assay is sensitive in detecting levels of ATP. The right panel shows the amount of ATP from mitochondria isolated from cultured human OSCs. Approximately 100,000, 10,000, 1,000, and 100 cells were lysed and used for analysis, with values and ability g in the mM to M range. Samples containing as few as 100 OSCs produced as much as 08 M ATP (about 600pmol ATP/cell). Compared to ovarian somatic egg cells, OSCs produce greater than or equivalent amounts of ATP/cell with approximately 100 fold less mitochondria.

Example 9: FACS-based Isolation of Mitochondria As described in this Example, ased s can be employed to e the total mitochondrial population in a . In addition, FACS-based methods for mitochondrial isolation can employ dual-labeling using two different fluorescent dyes (mitochondrial membrane potential (MMP)-dependent and dependent) to isolate only the fimctional (e.g., actively respiring) mitochondrial population or quantitate the ratio of functional to total mitochondria in a tissue, cell, lysed cell or fraction derived thereof.

The non-oxidation dependent acker Green FM (Invitrogen, Life Technologies Corp., Carlsbad, CA; M7514) mitochondrial tracking probe, which indicates mitochondrial mass, was ed and utilized as described below. MitoTracker stock solution (1 -5mg/m1 dissolved in anhydrous dimethylsulfoxide (DMSO)) was diluted in serum free growth medium to_reach a working concentration ofbetween 25-500nM. Freshly isolated or thawed OSCs were ed by centrifugation at 300 x g for 5 minutes. The supernatant was aspirated and the cell pellet was resuspended in 200 pl of the diluted MitoTracker stock solution.

Cells were incubated at 37° C for 45 minutes, washed in pre-warmed (37° C) serum free grth medium and pelleted by centrifugation at 300 x g for 5 minutes (alternatively, cells can be lysed prior to incubation with a probe of interest). Supernatant was ted and cells were resuspended in 100 pl mitochondrial lysis buffer and transferred to a FACS sort tube for lysis by mechanical permeabilization using rapid osmotic shock. Following lysis, cells were equilibrated on ice for 15-30 minutes, incubated in 200 pl (minimum ) ice cold PBS and vortexed. As shown in Figure 19, three ct populations were observed: residual M7514 positive cells (Cells MT +), high fluorescent mitochondria (functional, Mito MT high), and low expressing mitochondria (non-iimctional, Mito MT Low). The ratio of fimctional to non-fimctional mitochondria post lysis was approximately 1:1 (1552 mitochondria, 743 were gated as functional and 716 were gated as non-functional, with the remainder not gated; the game for each population ofmitochondria are highlighwd in Figure 19).

Therefore, fimctional mitochondria can be sorted and collected, with residual unlysed cells and non-fimcfional mitochondria excluded based on size and fluorescence intensity.

Dual- labeling using multiple probes or a JC-l probe (red spectrum Invitrogen, Life Technologies Corp., Carlsbad, CA; T3168) can help to fitrther guish flmctiOnal from mctional mitochondria. Probes for use in dual ng include, but are not limited to, reduced oxidative state mitotraclwr probes (c.g., ‘iacker Red CM-HZXRos (IIWih'ogen, Life Technologies Corp., Carlsbad, CA; M7513), MtoTraclaer Orange CM-HZMos (Irrvitrogm, Life Technologies Corp., Carlsbad, CA; M751 1) and accumulation dependent probes: JC-l (red spectmm; Invitrogen, Life Technologies Corp., Carlsbad, CA; T3168), MitoTracker Deep Red FM (Invitrogen, Life logies Corp., Carlsbad, CA; M22426) and JG! (green spectrum; Inviirogen, Life logies Corp, Carlsbad, CA; T3168).

Example 10: Mitochondrial IsolationUsing Differmtial Centl'lfhgation As described in this Example, difi'crential ccnlrifilgnfion Procedures can be yed to isolate sndlor fiactionatcmitochondriapresentin e. y steps Whenisolating mitochondriafi'om any tissue or cell are: (i)mpturingofcclll bymechanical and/or chemical means, (ii) difi‘erential cmhifiigation at low weed to remove debris and entemely large cellular lles (SPIN 1), and (iii) ccntrifugation at a higher speed to isolate and collect mitochondria (SPIN 2).

The tissue is d and washed twice with 1.5 ml of a Commercially available Wash Bufi‘er ences,_Abeam, plc, Cambridge, UK). The tissue is minced and placed in spre- chilled Dounee homogmizer. Up to 2.0 ml ofa commercially available Isolation Bufi'er (MitoSeiences, Abcam, plc, Cambridge, UK) is added. The cells are ruptured using the Dounce homogenizer (10-40 strokes), and the homogenate is transferred to Eppendorftubes. Each tube is filled to 2.0 ml with Isolation Bufl'er. The homogenate is cemrifisged at 1,000g for 10 minutes at 4‘C. The supernatant is reserved and transferred into new tubes, each ofwhich is filled to 2.0 ml with Isolation Buffer. The tubes are centrifuged at 12,000g for 15 minutes at 4'C.

The pellet is ed. Ifdcsired, the supernatant is analyzed for quality. The pellet is washed twice by resuspending in 1.0 ml ofIsolation Buffer supplemented with 10 p1 ofa commercially available protease inhibitor cocktail (MitoSeiences, Ahcam, plc. Cambridge, UK). The tubes are centrifuged at 12,000 g for 15 minutes at 4°C. Afierwashing, the pellets are combined and cnded in 500 pl ofIsolation Buffer supplemenhd with protease inhibitor cocktail. If desired, aliquots are stored at -80'C until use.

In one approach, mitochondria integrity is tested by Western blot screening for cytochrome c, porin, or hilin D in the isolated mitochondria versus in the supernatant traction using cially available antibodies, such as antibodies MSA06, MSAOS, MSA04 MitoSciences, Abcam, plc, Cambridge, UK). In another approach, ondrial samples are probed by Western blot to detect components of the mitochondrial complex, for example, using the commercially available OXPI-IOS Complexes Detection cocktail (MitoSciences, Abcam, plc, Cambridge, UK ). e 11: Mitochondrial Isolation Using Sucrose (instant Separation The protocol employs the following reagents, which are cornmemially ble: dodecyl-B-D-maltopyranoside (Lauryl maltoside; M8910; MitoSciences, Ahcam, plc, Cambridge, UK), ate buffered saline (PBS), Sucrose solutions 15, 20, 25, 27.5, 30 and 35 %, double distilled water, a protease inhibitor cocktail (Mitosciences, Abcam, plc, dge, UK ), and 13 r 51 mm polyallomer centrifuge tubes (Beckman 326819; Beclmian-Ooulter, Inc., Brea, CA).

The sucrose gradient separation procedure is a protein subfraclionation method optimized for mitochondria. This method resolves a sample into at least 10 fractions. It is le to separate solubilized whole cells into fiactions ofmuch lower couple-thy but when analyzing already isolated mitochondria the fractions are even more simplified. The sucrose gradient separation teclmique is designed for an initial sample volume of up to 0.5 ml at 5 mg/ml n. Therefore 2.5 mg or less oftotal protein should be used. For larger amounts, multiple gradients can be ed or larger scale gradients are made.

The sample is solubilized in a non-ionic detergent It has been determined that at this protein concentration mitochondria are completely eolubilioed by 20 mM n—dodecyl-fl-D- maltopyranoside (1% w/v lauryl maltoside). The key to this solubilieation s is that the membranes are disrupted while the previously membrane embedded multisubunit OXPHOS complexes rennin intact, at step ary for the density based sucrose separation procedure described herein. One important exception is the pyruvate dehydrogenase enzyme (PDH). In order to isolate PDH at a protein enation of 5 mgfml mitochondria, the required detergent concentration is only 10 mM (0.5 %) lauryl maltoaide. The PDH enzyme should also be centrifuged at lower speeds, a centrifugal force of 16 000 gis maximum forthe PDH complete To a mitochondrial membrane suspmsion at 5 mgl’ml protein in PBS. lauryl maltoside added to a final concentration of 1 %. This is mixed not and incubated on ice for so minutes.

The e is then centrifuged at 72,000 g for 30 minutes. A Beckman Optima benchtop ultraoenuii'uge (Beclcman-Coulter, 1110., Brea, CA) is reconnnended for small sample volumes.

However, at a minimum a benchtop microfuge, on maximum speed (e.g., about 16 000 g) should suffice. After firgation, the supernatant is collected and the pallet discarded. A protease inhibitor cocktail is added to the sample, which is maintained on ice. until centrifugation is performed. In samples very rich in mitochondria the cytochromes in complexes III and IV give the supernatant a brown color, which is useful when checking the efi‘ectiveness ofthe following separation.

A discontinuous sucrose density gradient is prepared by lavering successive decreasing sucrose densities solutions upon one another. The preparation and cermifugation of discontinuous gradient containing sucrose solutions from 15-35 % is bed in detail below.

This gradient gives good separation ofthe mitochondrial OXPHOS xes s ranging .10 ficmZOOkDato IDOOkDa). Howeverthissebrp cmbemodifiedforthe separation ofa particular complex or for the separation oflarger amounts ofmaterial.

The gradient is prepared by layering progressively less denSe sucrose solutions upon one another; therefore the first solution applied is the 35 % sucrose on. A steady application of the solutions yields the most reproducible gradient. To aid in this application, a Beckmm polyallorner tube is held upright in a tube stand. Next a Rainin Pipetman 200 pl pipette tip is placed on the end of a Rainin Pipetrnan 1000 pl pipette tip. Both snugly fitting tips are held steadybyaelamp ndtheendofthe200 plpipette tipis allowedto make ccntactwiththe inside wall ofthe tube. Sucrose solutions are thenplaced inside the 1000 pl e tip and fed into the tube slowly and steadily, starting with the 35% solution (0.25 ml).

Once the 35% solution has drained into the tube, the 30 % solution (0.5 ml) is loaded into the tube on top ofthe 35 % solution. This procedure is continued with the 27.5% (0.75 ml), % (1.0 ml), 20 % (1.0 ml) and 15 % (1.0 ml) solutious, respectiVely. Enough space is leit at the top ofthe tube to add the 0.5 ml sample ofsolubilized mitochOndi-ia.

Once the sucrose gradient is poured discrete layers of sucrose are visible. Having applied the sample tothe top ofthe gradientthe tube is loaded into theretorverycarefully, and cent-litigation begins. All fugation procedures e a balanced rotor therefore another tube emitainingpreciselythe samernasl is generated. Inpracticethismeans 2 gradientsmustbe prepared although the second nt need not n an experimental sample but could contain 0.5 ml water in place ofthe 0.5 ml protein sample.

The polyallomer tubes should be cenuiiiiged in a swinging bucket SW 50.1 type rotor (Beckman—Coulter, Inc., Brea, CA) at 37,500 rpm (Relative Centrifitgal Force avg. 132,000 x g) for 16 hours 30 minutes at 4'C with an acceleration profile of7 and deceleration profile of 7.

Immediately after the run the tube should be removed item the rotor, taking great care not to disturb the layers of sucrose. When separating a sample rich in ondria, te colored protein layers may be observed. Most ofien these are x 1]] (500 kDa ~ brown color) imately 10 mm from the bottom ofthe tube and Complex IV (200 kDa — green color) 25 mmflow the bottom ofthe tube. In some circumstances additional bands can be observed. These are_the other OJG'HOS complexes.

For fraction collection, the tube is held steady and upright using a clamp stand. A tiny hole is uced into the very bottom ofthe tube using a fine needle. The hole is just big enough to allow the sucrose solution to drip out at approximately 1 drop per second. Fractions of equal volume are collected in Eppendorftubes below the pierced hole. A total of 10 x 0.5 ml fi'ections are appropriate, however collecting more fi'actions which are thus smaller in volume iI also possible (e.g., 20 x 0.25 ml fractions). The fi'actions are stored at — 80'C until analysis.

Collected fractions are analyzed to determine mitochondrial integrity using any , methods described herein (e.g., in Example 9, 10) orknown in the art.

Example 12: OSCI Exhibit Increased Mitochondrial Activity It has been reported that low mitochondrial activity is a feature of "stemnesl", as it has been observed in togonia, early embryo, inner cell mass cells and embryonic stem cells.

See Ramalho-Santos et a1.. Hum Reprod . 2009 (5):553-72. 0505 are essentially the female lent of male spermatogonial stem cells (spermatogonia), however, it has now been determined that OSCs have prolific mitochondrial activity.

Following 080 lysis, mitochondrial production ofATP (pmol) was measured at 10, 15, and30 minutes, andthen standardized agairisttotalmtDNA content (fg)ineachsampletested (ATP Bioluminescence Assay Kit HS 11, Roche Applied Science, Mannheim, Grammy), As shown in Figure 20, adult human derived al stem cells , obtained from female patients between 22-33 (28.5 :i: 4.0) years ofage with Gender Identity er for reassignment at Sai’cama Medical Center, generated much greater levels ofATP than human mesmchymal stem cells fi'om bone marrow (hMSCs, obtained from PromoCell GmbH, Heidelberg, Gemumy), adult human ovarian somatic cells (subject d to the OSCs used), human embryonic stem cells (E805), and human d pluripotmt stem cells (iPSCs) derived fi-om lit/R90 fetal lung asts.

Mitochondrial production ofATP (pmol) was Itandardized t total mtDNA content (fg) in each sample tested. AI shown in Figure 21, ondria isolated fl'om adult human ovary-derived oogonial stem cells (OSCs) produced greater than 6-fold more ATP in 10 minutes than human hyrnsl stem cells (MSCs) from bone marrow and over 10-fold more ATP in minutes than adult human ovarian somatic cells (subject matched to the 0805 used), human embryonic stem celll (ESCs), and human induced pluripotent stem cells (iPSCs) derived fiom IMRQO fetal lung fibroblasts. Figure 21 depicts 1.03E-09, 1.46E-10, 1.76E-11, 4.56E-12, 9.10E— 11 pmol ATP generawd in 10 minutes for hOSC, hMSC, Soma. hESC, and hiPSC, respectively.

Standard errors (in the same order) are 1.15E—10, 4.56E-11, 2.28E-12, 1.72E-13 and 7.99E-12.

Deletion is revealed the presence ofthe common 49'?7-bp deletion in hMSCs (Figm‘e 22). Human ovarian coma, which is known to have the mutation, is included as ve control along with a no sample control (vs). The intact portion ofthe product was not detected in either . By comparison, the cornmnn p deletion is not detectable in hmnan OSCs (Figure 15).

Other ments From the foregoing description, it will be apparent that variations and modifications be made to the invention described herein to adapt it to various usages and conditions. Such ments are also within the scope ofthe following claims.

The recitation ofa listing ofelements in any definition of a variable herein definitions ofthat variable as any single element or combination (or subcombinafion) oflisted elements. The recitation ofan embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

This application contains subject matter that may be related to U.S. provisional application Ser. No. 61/502,840, filed June 29, 2011 and U.S. provisional application Ser. No. 61/600,529, filed February 17, 2012, the entire disclosures ofwhich are incorporated herein reference. All patents and ations mentioned in this specification are herein incorporated byreferenee to the same extent as ifeach indepmdent patent andpublication was specifically ' and individually indicated to be incorporated by rei'erence.

Claims (13)

1. A method ofpreparing an oocyte for in vitro fertilization (IVF) or artificial insemination, the method comprising transferring a composition comprising i) al stem cell (OSC) mitochondria, or ii) mitochondria ed from a progeny of an OSC, the OSC having been isolated from n tissue, into an autologous , thereby preparing said oocyte for IVF or artificial insemination, wherein the OSC is an isolated non-embryonic stem cell that is mitotically competent and expresses Vasa, Oct-4, Dazl, Stella and optionally a stage-specific embryonic antigen. 10

2. A method for increasing the ATP-generating capacity of an oocyte, said method comprising the steps of: a) providing a composition comprising mitochondria obtained from at least one OSC or at least one progeny of an OSC that is autologous to the oocyte, the at least one OSC having been isolated fiom ovarian tissue; and 15 b) injecting the ition comprising mitochondria into the oocyte in vitro, n the OSC is an isolated non-embryonic stem cell that is mitotically competent and ses Vasa, Oct-4, Dazl, Stella and optionally a stage specific embryonic antigen.

3. The method m 1 or claim_2, n the composition comprises 1x103 to 5x104 20 mitochondria.

4. The method ofclaim 1 or claim 2, wherein the OSC or progeny of an OSC produces at least 5-fold more ATP per fg mtDNA than an ovarian somatic cell or mesenchymal stem cell. 25

5. The method of claim 4, n the ovarian somatic cell or mesenchymal stem cell is autologous.

6. The method ofclaim 1 or claim 2, wherein the OSC or progeny of an OSC has been isolated from a human female (a) of advanced maternal age_and/or (b) with low ovarian reserve.

7. The method of claim 1 or claim 2, wherein the composition comprises mitochondria that have been isolated by centrifugation.

8. The method ofclaim 1 or claim 2, wherein the composition comprises mitochondria that have been isolated by mitochondrial membrane potential-dependent cell sorting.

9. The method of claim 1 or claim 2, wherein the ition is (a) a purified preparation ofmitochondria from the OSC or the progeny of the OSC or (b) the cytoplasm of the OSC or the progeny ofthe OSC without a nucleus.

10. The method ofclaim 1 or claim 2, wherein the composition is erred by injection into the oocyte.

11. The method of claim 2, wherein the oocyte is for use in in vitro fertilization. lO

12. An unfertilized oocyte prepared by a method as defined in any one of claims 1 to 10.

13. An unfertilized oocyte prepared by a method as defined in claim 11.