AU2015225014B2 - Feeder-free method for culture of bovine and porcine spermatogonial stem cells - Google Patents

Abstract

The present invention relates to the production and culture of undifferentiated spermatogonial stem cells that can be maintained long term and are feeder free. The resultant feedertee populations can be used in any of a number of protocols including the generation of progeny bulls, The present invention includes novel methods required for the successful enrichment of bovine spernatogonial stem cells, novel cell lines and other components used for the same, as well as the resultant stem cell compositions.

Description

ο (N α (D GO

O in (N (N in o (N :io in

TITLE: FEEDEE-FREE METHOD FOR CWLTURE OF ΒΟΤΙΝΕ AND: ΡΟΚαΝΕ SPiRMATOGONTAL STEM CELLS : applicationJs a divisionsl of Aastralian.Application Mo. 2Q| 3221S39, ihe ontiro COo&nts of which are incorporated herein by reference.

CROSS REFEREMCE TO RELATED APPLICATION

This appiieaiion claims priority under 35 C.S.C. § 119 to provisional application

Serial No. 61/598,437 tiled February 14, 2012, herein incorporated by rofererice in its entirety.

GRA.NT REFERENCE

This invention was triacle with government support under Contract No. 117232--awarded by the United States Department of Agrieultiire. The govemnlent has cetlair rights in the invention. 15 background of the invention

Sient cel is are undifferentiated cells that possess two hallmark properties; self-renewai and the ability to differefttiate into one or more different cell lineages. The prOGCSs of seif-renewal i nvolves the seif-replication of a stern cell to aHo w'for propagation and expansion, wherein the stem cell remains in an andilFerentiated state. Progenitor cells are 20 also undifferentiated ceils that have the ability to differentiate into one or more cell lineages, but have limited or no ability to self-renew. When maintained in culture, imdifferentiated cells, such as stem or progenitor ceUSi, can undergo spontaoeous differentiation, thereby losing the desired, undifferentiated cell phenotype. Thus, eniturc methods that minimize spontaneous differentiation in order to maintain the imdifferentiafed 25 stem or progenitor eel! state are needed.

Keeping iindiiferMtiated cells in an undifferentiated state is critical to their use, c.g., in industry and medicine, since a major scientitie and therapeutic usehilness of these ceils lies in their ability to expand into homogenous populations that can further proliicrate or differentiate into mature cells as needed, e.g., for scientific study or to repair damage to 30 cells or tissues of a paiieni. Once they have spontaneously differentiated in cell culture, the ceils are less proliferative and less able to differentiate into different types of cells as

Tigeded. A homogerious culture of undifferentiated steui eelis is theTefore a highly sought after but unrealized goal of research seientists and industry-

ο (N α (D GO

O (N (N O (N

Current methods for culturing undifferentiated oeiis (e.g., variotis tj/pes of stern eelis) attempt to minimize such sponianvous differentiaiion by delivering fibroblast grov^fh 5 factor 2 (FGF2) to the cell cultures daily, or, less frequently than every day, which is known as ’’feeding". FGF2 has been shown to proihote self-renewal of stem cells by inhibiting differentiation of the stem cell; however this inhibition is incomplete, and the stem Cell cultures tend to gradually difierentiate, thereby diminishing usefulness of the Stem cell culture. Furtheitnorej stem cells, such as embryonie or spermatogOnial stem ceUs, to typically need to be growm on mouse embryonic lbroblast :(idEF) feeder cells. This is a cumbersome step that is destrableto remove and results m a population of eel Is stem cells contaminated with the feeder cells that cannot be used in various fertilizatto protocols and production of gametes.

The ability to conditionally induce the development of stem cell fines through the iS process of spermatogenesis in vitro for the produetion of gametes would provide a long- SGught-after technology for biomedical research, and animal breeding particularly if such protocols could be established for a variety of species. To date, most success has been achieved in rats and mice only, leaving larger mammals such as bovine, wdthout such advances. 20 The discovery' that stem cells residing within fractions of dissociated mouse and rat testis cells maintain their ability to regenerate spermiatogenesis in testes of reeipient mice was essential to establishing such culture systems. See Brinster et al, PrOe Natl Acad Sci USA 1994; 91:11303-11307; Brinster et al„ Proc Natl Acad Sci USA 1994; 91:11298-11302; Clothier et aLj Nature 1996; 381:418-421; Kanatsu-Shinohara et al., Biol Reprod 25 2003; 69:612-616; andNagano etal., Tissue Cell 1998:30:389-397. The ability to isGlate and experimentally manipulate these stem cells has opened new doors for research on spermatozoon dcyelopmcnt, assisted reproduction, cclMar therapy and genetics. See Nagano et al., Biol Reprod 1999; 60:1429-1436; Mahanoy et al.. Endocrinology 2Q00; 141:1273-1276; Mahato et al,, Mol Cell Endocrinol 2001; r78;57~63; Ogawa et al,, Nat 30 Med 2000; 6;29-34; Shinohara et al., PmcNatl Acad Sci USA 2006; 103:13624-13628; Zhang et al., J Cell Physiol 2007; 211 :]49-15B; K.azuki et al,, Gene Ther 2008; 15:617-624; Kanatsu-Shinohara et al., Cell 2004; 119:1001-1012; Kanatsu-Shindhara et ai., Proc

Nati Acad Sci USA 2006; 103;80l8-8023; and Hagano etaL, Proe Mat! Acad Sfei USA 2001; 98; 13090-13095. In view of this poteirtiai, protocols far isdlati^ pfopagating and genetically modiifying Mly innciionai rat and mouse speitnatogomal stern cells in culture have been established. See Ryu et a!., Dev Biol 2004; 224:158-170; I&mra et aLiDev Biol 5 2004; 269:393-410; Hamra et at., Proc Matl Acad Sci USA2002; 99:;14931-14936; Hamia

ο (N Λ CD GO

O in (N (N O (N et al.j Methods MOl Biol 2008; 450:163-179; Hamra et al., PmcMatl Acad Sci OSA 2005; 102:17430-17435; Ryuet ai„ ProcNatl Acad Sci USA2005; 102:14302^4307; Orwig et al., Biot Repiod 2002; 67;874-879; and Ranatsii-Shinohara ei al.CBiol Reprod 2008. The mouse and mt were chosen as species for these studies due to their popularity as taboratory i p animal models for the study of human health and disease, and due to the laclE of protocols for genetically modifying the rat germlinc using elonaliy c.xpanded stem cells from culture. See Hamra et al, Proe Natl Acad Sci USA 2002; 99:14931-14936, Gonsidering the inany potential applications of the laboratory rat as a research model, a cOst-efFective and easy-to-prepare culture medium was souglit in this study for the derivatioh and eontiniious 15 pro iifcratioh of primary rat spermatogonial stem c eil lines in vitro.

Despite tl'iese advances, even in the rat species^ the proeedure remains complex and largely unsueeessful. For example, media for iohg-teim proliferation of rodent spermaiogoniai stem cells in vitro are relatively Cornplex, expeftsive, time-consurhing to prepare, plus are most effective when applied in combination with feeder layers of 20 fibroblasts.

As can be seen a need exists for methods of culturihg spennatogonial stem cells, particularly for larger mammals such as bovines.

SUMMARY OF THE INVENTiON 25 The present invention comprises compositions and methods for culturing spematogonial stem cells. According to the invention, Applicants have developed a fbeder-free culture system that allows for cultured spermatogonial stem cells to remain in an undifferentiated state and to remain viable in culture for long periods of time. Further: aspects of the invention include procedures to isolate undifferentiated spermatogonia from 30 testicular tissue, the identification of speeifr e serum-free media that supports survival of undifferentiated spermatogonia In vitro, substrates for coating of plastic culture wells that undifferentiaied spermatogonia adhere to, and finally Applicants have identified specific

ο (Ν <D GO Ο ΙΟ (Ν (Ν ΙΟ Ο (Ν ΙΟ @'owth factors for the sappiementation of feeder cell eonditioiied media that promotes maintenance and growth of bovine nndifferehtiated speiTnatogoinia. Whil e the methods and examples disclose borne cells, the invention is not so limited and is applicable to all livestock species including poreme. 5 According to the invention. Applicants have identified specific proprietary eell lines including a hovme mnbiyottie fibroblast cell line, and a bovine somatic eel! line isolated from bovine testis. These specific cell lines are used as feeder support cells to condition culture media prior to eultivation of any SSCs, The pre-eultivation technique allows for feeder free cultivation enabling the generation of pure populations of putative 10 bovine SSCs for transplant or other uses. Applicants’ eiiltitred cells are free firom contamination with other feeder cell lines and their deleterious effects, including poteniial host immune response, limited eoloniring ability, and eventual differentiation over time.

In an embodiment, the present invention foatorcS a method of isoiating undiflerentiated spermatogonia, of which spermatogonial stem cells (SSCs) are a 15 component, from testicular tissue containing at least one SSG. The method includes obtaining bovine testicular tissue which includes at least one SSC, eontaeting said tissue with collagenase, separating seminiferous tubules from other cell types, and thereafter eontaeting said seminiferous tubules with trypsin to yield a cell suspension enriched in spermatogonia and Sertoli cells. The method may also be applied to SSCs generated Ifom: 20" induced piuripotent stem cells Or even embryonic stem eells that have been manipulatedfo beSSCs.

In another embodiment, the invention features a method of enriching and maintaining a pure culture of imdifferentiated spermatogoniai cells contaming at least one SSC, wherein the metltod includes providing a specific semm-fi-ee media with serum 25 replacement:, further providing culture cell wells that are pre-eoated with Matrigel, and adding speeifie gfowth factors of GDNF, PCFS, SE)F-1, and CSF-1 to pre-conditioned media all work together to provide for the first time a feeder-free culture of spermatogonial ceils, preferably bovine spermatogonial cells.

In an embodiment, the invention also features a putative population of 30 speimatogoniai stem eells that remains undifferenfiated, possesses the capacity for self-renewal and differentiation, is vdable in culture, and is esseBtially pure, namely feeder-free. ο (N α Ό in ο ΙΟ (Ν (Ν ΙΟ Ο (Ν IT)

ίη another embodiment the invention includes novel support cell lines which have been developed and which allow for the pre-conditioning of media to provide for the feeder tree culture method. The cell lines include ceil line bovine embiyonic fibioblast 1 (or BEF1, previously BEF) derived froin a 35 day old male Holstein fetus, and the sccohdvccil 5 lines bovine somatic cell 1 (or BSG1, previously BSGFderived ihbin somatic cells isolated fi-om the testis of a 4 month old Holstein bull. Together these cell lines may be used to pre-meUbate culture media and will provide neecssary secreted soluble factors for suivdval and proliferation of SSCs without the addition of feeder ceils once the SSG have been introduced, According to the invention the media is first pre-eonditioned with these cells 10 and after a sufficient period of time, the feeder precondition cells are removed so that SSG cells may then be cultured.

In one aspect of the invention, the SSG is a bovine SSG. In another aspect, an SSG is derived irom an organism seieeted from the group consisting of a mouse, a rat, a monkey, a baboon, a human, a pig and a dog. 15 in another aspect of the invention, cells are derived from ahy source of

Spermatogonia! stem cells including a source selected fi-om the group consisting of wild type adult testis, calf or pup testis, neonate testis, and cryptOrchid adult: testis.

In another aspect of the invention the ceils are derived fiOm induced piuripotent stem cells or embryonic stem cells to become spermatogonial stem cells, which then may 20 become a source of the same used according to the invention.

In another embodiment the invention featiires a feeder-fice culture system for support of SSG maintenance, the system comprising enriched SSCs, serum-fiee defined culture medium, and media precOhditianed with feeder cells. In another embodiment, the invention features a feeder-free culture system for siippoft of SSG proliferatiGn comprising 25 at least one SSC, serum-free defined culture medium with serum replaGeraent supplement (StemPro), and which has been preconditioned w'ith the pfoprieiary fibioblast cell line BEFl and proprietary BSC 1 feeder cells.

In one aspect of the invention, a cultnre system, further comprises growth factors of GDNF, FGF2, SDF-1, and GSF-1, M another aspect, a culture medium comprises at least 30 one medium seieeted from the group consisting of Dulbecco's MEM: Flam's Nutrient Mixture F-12 (PMEM/Fl 2) and StemPro serum replacement .supplcinent. ϊη another aspect of the invention, a culture systetn farther copprises g CTiltiire wells that are pre-coated with a eommercially available matrix such &amp;vS Matfigei (growth factor reduce-d version). Without such coating the cells do hot attaeh to the p: well and cannot be maintained long-tenn in culture.

ο (Ν α (D GO 5 In another embodiment^, the invention features a composition comprising a Ο (Ν (Ν Ο (Ν population of pure, enriched S SCs, vvherein the enriched population of SSCs is free of embryonic fibroblast cells or other tyjres of feeder cells.

In another embodiment, the invention features a compGsition eompri.smg a population of enriched SSCs which expiess a specific marker of undifferentiated to speimatogoma and a morphology that is similar to that of cultiired SSC cells. In one aspect, the population of SSCs is substantially homogeneous for SSCs.

In yet another embodiment the mventiOn features a method of generating at least one mammalian pfogehy, comprising administering a population of feeder free SSCs to a testis of a male recipient mammal, allowing the enriched SSCs to generate a colony of 15 spermatogenesis in the recipient mammal, and mating the recipient mammal with a female mammal of the same species as the recipient mammal. In one aspect, a population of enriched SS Cs is administered to the lumen of a seminiferous tubule of the recipient mamrnal. In another aspect, the recipient mammal is infertile.

In an embodiment of the invention, a recipient mammal is seleeted fix)ni the group 2G i consisting of a rodent, a primate, a dog, a bovine, a pig and a human. In another etTibodiment j a rodent is selected from the group consisting of a mouse and a rat. In yet: another aspect, the primate is a baboon.

In another embodiment, the invention features a kit for maintaining at least one SSC in a feeder^free culture system. The kit includes a culture system Gomprising semm-25 free defined culture medium, proprieiary BSCl and BEFl cells for pre-conditioning eulture medium, (or medium that has already been preconditioned with the same), an applicator, and instruetional material, wherein the instructional material comprises instructions for the use ofthe kit to maintain at least one SSC in the feeder-free culture system.

In an embodiment, the inventioh features a progeny animal produced according to a 30 method ofthe inventidn, in another embodiment, the invention fethum^ progeny animal made according to a method ofthe invention, vdiemitt the enriehed SSCs use^ make the 6 progeny animal contaitt at least one genetic rmttaiion. In one aspect, a gonetic mxitat^ created using recombinant techniques.

ο (N Λ CD GO

The ciaimed invention comprehends, in part, using certain natumlly occurring or transgenieaily generated ("genetically") male-sterile hovines as recipients for donor 5 spermatogoniai .stem cells with w'hich the animals also are immuno-eompatible.

O in (N (N in o (N

Accordingly, the transplanted spermatogoniai stem cells are free to develop into functional spermatozoa and to fertilize females in the absenceof competition #om sperm that also would be produced were the recipient male feitile. in this manned 100% germline transmission of the donor cell haplotype can be achieved from a relatively low number of 10 transplanted sperm stem cells.

Thus, pursuant to one aspect of the present invention, a methodology is provided for effecting germiine transmission of a donor haplotype. The inventivemethod comprises the steps of (A) providing cells of a spermatogoniai stem cell line that is derived from mammal testes, from induced pluripotent stem cells, or embryGiniuc stem cells, v/hich Gell 15 line embodies a predetermined genetic backgronnd, and then (B) tj-ansplanting one or more of the cells into a male-sterile recipient, such that transplanted cells develop into fertilization-competent, haploid male gametes.

In accordance with andthef aspect of the inventiGri, a library is provided of cells of a spermatogoniai stem cell line that is derived fi'om mammaitestes, or from induced 20 pluripotent stem cells, or embryoiniue stem cells. A library of the invention contains a pluraiity of gene knockout or "Mockin" mutant stem, cell^ v

DESCRIPTION OF THE FIGURES

Figure 1 shows the Maintenance of bovine nndiffereritiated speiTnatogonia in 2.5 feeder-free culture. Left image is representative of a gerni cellclump formed during feeder-free culture on Matrigel coated.culture wells: with BIFI conditiohed media. The middle image is the Glumps stained with D API tptahcl nuclei of individual cel Is, The right image is immunostaining for the undifferentiated spenMatogbnial marker PLZF.

30 DETAILED DESCRlPTiON OF THE INVENTION

Unless otherwise defined herein, scientifte and fsehmeal terras Used in conncGtion with the invention shall have the meanings that are commonly understood by those of 7 ordinary skill iri the ait. Further, unless otherwise required by eotitext, sin:plar tertns shall include- the plural and plural terms shall include the singular. Generally, nomeiiclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and celiular biology, microbiology, genetics and protein and nucleic acid chemistry and 5 hybridization described herein are those well known and commonly used in the art. d'he methods and techniques are generally performed accordihg to eonventipnal methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al Molecular Gldtiing; A Laboratory Manual, 2d ed., Cold Spring 10 Harbor Laboratory l?ress, Cold Spring Harbor,iN.y. (1989); Ausubel et al, GUirent

Ο (N Λ CD GO

O lO (N (N O (N

Protocols in Molecular Biology, Greene Publishing Assoeiates (1992, and Supplements to 2002); Harlow' and Lane, Antibodies: A Laboratofy Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor and Dricfcamer, Introduction to Glycobioiogy, Oxford Univ, Press (2003); Worthington E:nzyme Manual, Worthington I s Biochemical Corp., Freehold, N.J.; Handbook of Biochemistry:; Section A Proteins, Vol, I, CRC Press (1976); Handbook of Biochemistry; Section A Proteins, Vbl. II, CllC Press (1976) ; Essentials of Glycobioiogy, Cold Spring Harbor Laboratory Press (1999).

The following terms, unless otherwise indiGated, shall be uUdefStood to have the following meanings: 20 The phrase "spermatogonial stem cells*' in this description denotes stem eelis either isolaicd from the testis, created from induced pluripotent stem cells, from emhiyonic stein cells or any other method to obtain such cells. For example, maffiinaiian indueed pluripotent- stern cells created from skin cells haye been used to ereate geim cells. See, Easley CA 4* etal, CelLRep. 2012 Sep 27;2(3):44θ-6, “Direct differentiation of human 25 pluripotent stem cells i nto haploid spermatogente eelis”. SpermatogOnial stem cell s are incapable of foitilizing an egg celibut can give rise to cells that deyelop info sperm and so produee viable ofrspring- ISOlaied spermatogonial stem cells can be cultured: for a prolonged time period without losing their properties and can eifrciemiy mpopulate the testes of suitable recipient male animals described, for instance, in Gatley j, M. et aL, 30 Methods Eazymol, 419:259 (2006).

The articies "a" and "an" are uaed herein to refer to one or to more than one (i.e,, io at least one) of the grai’nmatieal object of the artieie . By rvay Of example j ''an eletrien t" means one elernent or more than one element.

Ο (N α CD GO

O in (N (N in O (N

As nsed herein, the teims "gene" and "recombinant gene" refer to nttcleic aeid 5 raoleeules comprising an open reading frame encoding a polypeptide. Such natural allelic variations can typically result in 1-5% variance in the nucleotide .sequence of a given gene. Aitemative alleles can be identified by sequencing the gene of interest in a number of different individuals. This can be readily carried out by using hybridization probes to identify the same genetic locus in a variety of individuals. .Any and all such nucleotide 1:0 variations and resulting amino acid polymorphisms of variations that are the fesult of natural allelic variation and that do not alter the functional aetivity are intended to be withiu the scope of the invention.

Moreover, nucleic acid molecules encoding pfoteins from other species (homologs), wMch have a nucleotide sequence which differs from that of the proteins deseribed herein IS are within the scope of the invention. Nucleic acid: molecules corresponding to natural allelic variants and homologs of a cDN A of the myentioa can be isolated based on their identity to nucleic acid molecules using the human cDNAs, or a poriion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions. 20 As the term is used herein, "modulation" of a: biological process refers to the alteration of the normal course of the biological process. For example, modulation of the aetivity of a spermatogonial .s tem cell may be an increase in the activity of the cell Alternatively, modulation of the activity of a spermatogonial stem eel} m ay :be a decrease in the activity of the cell 25 "Enriching," as the term is used, herein, refers to the process by whieh the concentration, number, or activity of something is increased from a prior state. For example, a population of 100 spermatogonial stem cells is eottsidered to be "enriched''in spermatogonial stem cells if the population previousiy contained only 5:0 spermatogonial stem cells. Similarly, a population of 100 spermatogcarial stem cells is also considered to 30 be "eHriched" in spermatogonial stem eells if the population previousiy contained 09 spemtatogonial stem: ceils. Likewise, a populalion of iOO spermatogonial stem: cells is also ο (N α aj in

o in (N (N in o (N in eonisidered to be ’'emiehed" in spermaiogoniai stem cells even if the population prevloiisly contained zero spermatogonial stem ceils.

As the tenri is used herein* "population” refers to two or more cells.

As the term is used herein, "substantially separated from” or "substaniially 5 separating" refers to the characteristic of a population of first substances being removed fiom the proximity of a population of second substances, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessariiy devoid of the first substance. How'ever, a population Of first substances that is "substantially Separated from" a population of second substances has a 10 measurably lower content of second substances as compared to the non-separated: mixture of first, and second substances.

In one aspect^ a first Substance is substantially separated from a second substance if the ratio of the conGentralion of the first substance to the coneentration of the second substance is greater than about 1. In another aspect, a first substance is substantially 1 .s Separated fiOm. a second substance if the ratio of the concentration of the first subStanee to the concentration of the second substance is greater than about 2. In yet another aspect, a first substance is substantially separated from a second substance if the ratio of the concehtration of the first substance to the concentration of the second substance is greater than about 5. In another aspect, a first substance is substantially separated from a second 20 substance if the ratio of the coneentration of the first sUbstance to the «meentration of the second substance is greater than about 10. In still another aspect, a fihst smbstance is substantially separated from a second substance if the ratio of the concentration of the first substance to the concentration of the second substance is greater than about 50. In another aspect, a first substance is substantially separated ixom a second substanee if the ratio of 2.5 the coneentration of the fi rst substance to the coneentration of the second substance is ^eater than about 100. In still another aspect, a first Substance is substantially separated from a second substance if there is no detectable level of the secohd substance in the composition containing the first sub.staTiGe. "Substantially bomogeneous," as the term is used herein, Fefrrs to a popidation of a 50 substanee that is eoinprised prirnarily of that subsianee, arid one m which impurmes have been minimized. 10 "Maintenance*' of a ceil or a population of cells refers to the eondition in whi ch a cell or living cell population is neither increasing nor decreasing iri total nurnher of cells in a culture. Alternatively, "proliferation" of a cell or population of cells, as the temi is used herein, refers to the condition in which the number of living cells increases as a 5 function of time with respect to the original number of cells i n the cul ture. Ο (N α (D in

o (N (N O (N A "defined culture niedium" as the teim: Is: used herein refers to a pell eultnre medium with a known composition,

Bythcterm ''applicator" as the tennis used herein, is meant any device including, but not limited to, a hypodermic syringe, a pipetie, a bronchoscope, a nebulizer, and the 10 like, for administering a composition of the invention to a mammal.

As used hefeinj an "instructionai material " inelude.s a publication, a recording, a diagram, or any other medium of expression which can be used to conimunicate the usefulness of a method and/or composition of the inventipn in a Mtfbr maintaining, proliferating, or administering any eomposition recited herein. Tne instructional material 15 of the kit of the invention may, for example, be affixed to a container which contains a cornpositionof the invention or may be shipped together with a container which contains a cornposition, Altematively, the instructional material may be shipped separately from the container with the intention that the instructionai material and the compound be used cooperatively by the recipient. 20 As the term is lised herein, a cell is said to he "eliminated" from a population of cells, Or from a culture medium, when the cell no longer exerts one or more ofa physieat, biological or ehemical effect on the population of cells or culture medium. For example, a ceil may be eliminated from a culture medium by physically removing fee cell using FACS or by using an antibody speexfie for a cell surface marker unique to that celi, A cell may 25 also be eiiminated from a culture medium by rendering the hiological aettvity of that cell inert, such as, for example, by using a nfixpralizing antibody that is specifre fer that cell, A ceil is ''essentially eliminated" from a population of cells, or froin a eulture medium, wixM most, but not all of the total number of such ©efisno longer exerts one or^ more of a physical, biological or chemical effect on the population of cells or culture 30 medium. For example, a particular type of cell may be essentially eliminated from a culture medium if at lea.st 75% of the cells of that type are removed from the culture rnedium by using an antibody specific for a cell surface nxaiker unique to that celi More 11

Ο (N α (D GO

O in (N (N in H O (N in preferably at least 80% of the cells are eliminated fix>m the culiiiremediam, even more preferably at least 85%, rriOre prefferably, at least 90%, and eveti more preferably, at least 95% of the ceils are elimmated iTom the cviliure medium, A ceil is a "testis-deriYed” ceil, as the term is used herein, if the eeil is derived from 5 a testis. By way of a non^iimititig exarnple, testis-derived ceils iheiude a spermatogonial stem cell, a somatic cell, and a germ: cell.

Methods of Enriching Spermatogonial Stem Cells

The present invention features a method of enriching spermatogonial stem cel is 10 fSSGs). It has been shown for the first time herein that, SSGs may be cultured in a feeder free protocol with long term survival to enrich a population of cells. In one embodiment of the mvention, an SSC is obtained from a bovine. Stem ceil enriehment is useful for various purposes in the field of medical treatment, diagnosis and research, including stem-cell based therapies for repopuiation of the ceils in an organism, as well as laboratory researtdi I S to identify growth factors responsible for control of foe maintenance and proliferation of stem cells as well as for artificial insemination.

The present invention comprises eorapositions and methods for culturing spematogonial stem cells. According to the invention, Applicanm have devefoped a feeder free culture system that allows for isolated (or othenvise ohtained) bovine: spgfj^iaiQgomal 20 stem cells to remain in an undifferentiated state and to remain viable in culture for fong periods of time. Induced piuripoient stem cells or embiyonic stem cells eou Id also be used. Further aspects of the invention inel ude procedures to i splate undiffereniiated spermatogonia from bovine testicular tissue, the identification of specific serum-free media that supports survival of bovine iindifierentiated spermatogonia in vitro, substrates for 25 Coating of plastic culture wells that cultured bovine undifferentiated spermatOgOnia adhere to, and finally Applicants have identified specific growth factors lor the supplementation of feeder cell conditioned media that promotes maifttehance and of bovine tmdifferentiated spermaiogoinia.

According to the invention. Applicants have identified specific proprietary cell 30 lines including a fibroblast bovine embryonic cell ΙίηΟ, and a boyine somatic cell line isolated from bovine testis or otherwise Obtained. These Specific ceil lines are used as feeder support cells to condition culture media prior to Gultivaiion of any SSCs. The pre- 12 eultivaiicm technicpe alio feeder free cultivatiDn enabling the genef^^ populations of bovine speimatogonial SSGs for tiansplant or other tuses without eontaminahon from other feeder cell lines and their deleterious effects ineluding potential host immune response, limiting the colonizing ability of the SSCs. ο (N <D ΟΟ 5 In an embodiment, the present invention features a method of isalating Ο (Ν (Ν Ο (Ν spermaiogonial stem eelis (SSCs) from bovine testicular tissue containing at least one SSC, The method ineludes obtaining bovine testicular tissue which includes at least one SSC, eontacting said tissue with collagenase, separating seminiferous tubules from other cell types, and thereafter contacting said seminiferous tubules with trypsin to >ield a cell IQ suspensiGn enriched in spermatogonia and Sertoli cells.

In another embodiment, the invention features a method of enricbing and maintaining a pure culture of spermatogonial stem cells (SSCs) from a population of testis·^ derived, iriduced pluripotent stem cells, or embryonie stem cells containing at least one :SSC, wherein the method includes providing a speeifrc serum-free media with serum: 15 :- ieplaecmeni, further providing culture cell wells that arc pre-coated with Matrigel, and adding specifxe growth factors of GDNF, FGF2, SDF-1, and CSF-1 to pre-conditioned media all work together to provide for the first time a feeder-free cultui’e of spermatogonial cells, preferably bovine spermatogonial ceils.

In an embodiment, the invention also features a population of spennatogoftial stem 20 cells that retnains undif&amp;reniiatcd, possessing the capacity for selfirenewal and differentiation, is viable in culture, and is essentially pure, narnely feeder-free:.

In another embodiment the: invention includes novel support cell lines which have: been developed and wbich allow fbrthepre-condiiionmg of media to provide a feeder free: culture method. The cell lines include cell line BEFl deiiyed from a 35 day old male 25 Holstein fetus, and the second, cell lines BSCl derived frofr! iSOmatic cells isolated frOm the testis of a 4 month old Holstein bull. Together these cell lines may be used to prerihcubate culture media and will provide necessary secretion factors for survival and proliferation of SSGs withQut the addition of feeder cells to the incubating SSCs,

In one aspect of the invention, ah SSC is a bovine SSC. In another aspect, an SSC 30 is derived from an organism selected from the group consisting of a mouse, a rat, a monkey, a baboon, a human, a pig and a dc 13

In another aspect of the invention, cells are derived from a source selected froni the group consisting of wild type adult testis, pup teStiSi, neonate testis, induced pl uripotent ceils, embtyonic stem cells, and^'cryptorchid adult testis.

Ο (N α (D GO

O (N (N O (N

In an embodiment, the invention features a serum-free culture system for support of 5 SSG mainienanCe, the system comprising enriched SSCs, serum-free deSned culture rnediuin, and media preconditioned with feeder cells, in another embodiment, the invention features a senim-fi-ee culture system for support of SSG prolifemtion comprising at least one SSC, serum-free defined cUiture medium with serum replacement suppiement (StemPro), and which has been preeonditioned with the proprietary fibroblast eel 1 line 10 BEFl and proprietary BSC 1 feeder cells.

In one aspect of the invention, a culture system further compiises growth factors of GDHF, FGF2, SDF-1, and CSF-1. In another aspect, a culture medium comprises at least one medium selected from the group eonsisfing of Dulbeceo's MEM: Ham's Hutrient Mixture F-12 (DMEM/Fl 2) and Stem-Pro serum replacement supplement, 15 In another aspect of the inventionj U culture system further comprises growth plastic :: culture wells that are pre-coated with a eommercMly available matrix ;sueb as Matrigel : (growth factor reduced version). Without such coating the ceils do not attach to the plastic well and cannot be maintamed long-term in culture.

In an embodiment, the invention features a composition comprising a population Of 26 i pure, enriched SSCs, wherein the enriched population of SSCs is free of embryonic fibroblast cells or otber types of feeder ceils.

In another embodiment, the invention features h compositioncomprising a population of enriched SSCs which expre.ss a speeific irtarker of undifferentiated spermatogonia and a morphology that is similar to that of eultured SSC cells. In one aspect, 25 a population of SSCs is substantially homogeneous for SSCs,

In an embodiment, the invention featuins a method of igenerating at least one mammaiian progeny, composing administering a population of feeder free SSCs to a testis of a male recipient mammal, allowing the enriched. SSCs to generate a eolohy of spermatogenesis in the recipient; mammal, and mating the recipient mammal with a female 36 mamma! of the same species as the recipient mammal. In one aspect, a population of enriched SSCs is administered to the lumen of a seminiferous tubule of the recipient mammal. In another aspect, the recipient mammal is infertile. 14 ο (N α ω m ο (Ν (Ν ΙΤ) Ο (Ν 10 1:5 25 Τη an embodiment of the inveTition, a recipient maipnnal is selieoied from the grdirp consisting of a rodent, a primate, a dog, a bovine,; a pig and a human. In another embodirnent, a rodent is selected from the group consisting of a tnotise and a rat. lin yet another aspect, the primate is a baboon. in one embodiment, the invention features a method of generating at least one progen y mammal, eomprising adrriinistering a population of feeder free enriehed SSCs to a testis of a male recipient mammal, allowing the enriched SSGs to generate a eolohy of spermatogenic ceils in the recipient mammal, and mating the recipient mamm^ with a female mammal of the same species as the recipient mammal.

In another embodiment, the invention features a kit tor mainiaining at least one SSC in a feeder^ffee culture system. The kit includes a culmre systern comprising seruin-ifee defined culture medium, proprietary BSCl and BEFl cells for pre-conditioning culture medium, an applicator, and instructional material, wherein the insiTuetional material comprises instruefions for the use of the kit to rnaintairi at least one SSC in the feeder-free culture

In an embodimeBt, the invention features a progeny animal produced according to a method of the invention. In another embodiment, the invention features a progeny animal i ihade according to a method of the mvention, wherein the enriched SSGs used to make the ΐ progeny animal contain at least One genetic ifiutatiGn. In one aspect, a genetic mutation is created using recombinant teehniques.

The claimed invention comprehends, in part,: using certain naturally oecuning or transgenicaliy generated ("genetically") inaie-sierile bovdnes as meipients for donor sperm stem cells with which the animals also are immuno-compatibie. Spermatogenesis in these animals is severely disrupted. Accordingly, the transplanted sperm stem cells arc free fo develop into functional spermatozoa and to fertilize females in the absence of competition from sperm that also would be produced were the recipients male-fertile. In this manner, 100¾ getm line transmission; of the donor cell haplotype can be achieved from a relatively low number of transpianted sperm stem: cells. ' Thus, pursuant to one aspect of the present invention, a methodology is provided for effecting geitnline transmission of a donor haplotype. The inventive method comprises the steps of (A) providing cells of a Sperinatogonial stem cell line that is derived from mammal testes, from induced piuripotent stem ceils, or created from embryonic: stein ceils 15 ^'hieh cell line embodies a predetermined genetic backgroiind, and then (B) transplantmg one or more of the eells into a male-^erile rec^ient, such that transplanted cells develop into fertilizatiGn-compeienti haploid male gametes.

ο (N Λ CD GO

O (N (N O (N

In accordance with another aspeci of the invention, a library is provided of cells of 5 a spennatOgonial stem cell line that is derived froin mammal testes, ind uced from pluripotent stem cells, or created from embryonic stem cells. Λ library of the invention contains a plurality of gene knockout or "knockin·' mutant stem Gells.

In accordance with an additional aspect of the invention, am.edium for grow'ing spermatogoniai stem celts is provided, in addition to methods for culturing spetmatogonial 10 stem ceils,

Feeder-Free Sperinatogonial Stem Cell Cultare Systems

The present irtvention features a feeder-free culture system for the in vitro maintenance and proliferation of SSCs. This is because it has been shown hemih: for the 15 ; first time that bovine undifferentiated spermatogonia, of which SSCs are a component of, i can be maintained and proliferated in media that has been pre-conditioned mth feeder ceils, but that does not contain feeder cells in vitro. By way of the present invention^ a culture system, having minimai, defined conditions has been estahlished for the in vitro culturing of j bovine SSCs, which system provides the ability investigate SSC biology in a defined way, 20 : as w'ell as the ability to identify individual factors required for maintenance and expansion of SSCs. In a preferred embodiment, the feeder fine culture method allows for generation of a population of SSC cells that is freefrom CGntamination by feeder cells for use in \titro fertilization and other aspects of commercial bovine production, in one embodiment of the invention, a feeder-free SSC culture system includes a 15 preconditioned feeder medium. In one aspect of the i nyention, the preconditiGning feeder cells are Applicants’ proprietary cell lines including the bovmeernhryenie fihiOhlasi^ BEFi and the bovine somatic cell line BSCl, In another aspeet of the mventiou, the preconditioning feeder eelis ean include, but are not limited to, eleinents derived from these lines, including cells, eornponents thereof, and the like. 30 Applicants have identified a critical medium for culture of the SSCs, This in one aspect of the invention, the medium for culture and preconditioning is a serum-fi’ee defined medium includes minimal essential medium-a (MEMo). In another aspect of the invention, 16 the sserurn-free defined mediiim inchides Ham’s FlO cnltdre tnedinffi. In yet another aspect of the inventidn, the serum-free defined medium is Didbecco^ MEM: Hanf s 'Nutrient Mixture F-12 (DMEM/F12), As will be understood by the shilled artisan when armed with the present disclosure, a serum-free defined medium of the present in vention also ineludes 5 a mixture of two or more media wherein one of which is DMEM/F12. ο (N <D C/5

O (N (N O (N

Therefore, the present invention ineludes a composition including a defined: medium that has been precoiiditioned with one or more feeder eeli lines for the maintenance or proliferation of SSCs. As will be understoodhased on the disclosure set forth herein, a culture system of the invention is usefid for the maintenantcc or expansion of 10 SSCs, In one aspect of the invention, SSCs useful in the eulture system are SSCs enriched using the methods or compositions of the present invention. In another aspect, SSCs useful in the culture system are SSCs that have not been previously enriched according to the methods or compositions of the present invention.

In one embodiment of the invention, a feeder-free defined medium further includes 15 :SSCs, The skilled artisan will understand, in view of the present disclosure, that SSCs > from any source may be maintained or expanded using the feeder-free culture system of the : invention. That is, SSCs obtained ft'ora a population of testis-derived ceils can be obtained from testis cells derived from any maminalian smtree including, but not linuted to, hiiman : testis, rat testis, mouse testis, and preferably, cow or bovine testes. Sources of SSCs 20 s further include wild type adult testis, induced pluripotent stem Gells, emteiymnie stem cells, adult testis having one or more genetic mutations, juvenile testis, neonate testis^ aniEor eryptorchid adult testiS: Methods for introduciion, of genetic mutations to the ©N A in a eell, such as an SSC, are well-known in the ait and will not be discussed further herem.

It vrill also be understood, based on the disclosure set forth herein, that a sterile 25 male can be a source for SSCs, Therefore, another aspect of the mvention includes a sterile male as a source of SSCs of the present invention.

As described in detail elsewhere herein, the basic feeder-free defined medium of the preseni inventiGn tnay further include any components known by the skilled artisan to be useful in the culturing of cells. In one embodiment, a feeder-free deihed mediura 30 includes at least one growth faeior. GiOwth factors uselui in the present invention inclufie, but are not limited to, stem ceil factor (including mouse SCF), glial cell line-derived neurotrophic factor (GDNF), GDNF-family receptor (ineiuding GFR,alpha. l leukemia Γ

Ο (N <D OO

O in (N (N in o (N in inhibitory factor (LIF), basic fibrobiast growth factor (including human bFGF), acidic fibroblast growth factor (aFGF), colony stmTulating factor (CSF), stromal derived factor 1 (SDF-1), epidermal growth factor (EGF), insuim-like growth factor (including IGF-f), platelcbdcriyed growth factor (PDGF), and transtorming growth factor (ihcludaig TGF-j3 I 5 through ill, as well as the TGF β. superfarnily BMP--1 through 12, GOF 1 through 8, dpp, bOA, ΒΪΡ, OF), in a preferred embodiment the growdh factots inelude GDNF, FGF2, and OSF-l.

The present invention therefore also includes methods of mamtairiing or proliferating SSCs in a feeder-free defined culture medium. In one embodiment, the 10 invention features a method of maintaining SSCs in a feeder-hee culture system. The method includes providing at least one SSC in a feeder-iree culture system as defined in detail eisewhere herein.

An SSG may be identified as being "maintained" in the fesder-tme dehned culture system by assessing the activiij' of an SSC at various time points in the culture medium and 15 toomparing the activity with the aetivity of the SSCs at the start of the euiture peiiod. As will be understood by the shilled artisan, little or no loss of activity is an indication that SSCs have been maintained in culture. Methods of measuring the activity of SSCs are described in detail elsewhere herein.

In another embodimentj, the invention icatures a method of proliferating S SCs in a 2:() feeder-free culture system. The method ineiudes providing at lea.st one SSC in a feeder-free culture system as defined in detail elsewhere lierein. A SSC may be identified as being ''proliferated" in the feeder-tree defined euiture system by assessing the SSC activity at various time points in the culturing process and comparing the aetivito vvith the activity of the SSC at the stait of the culture period, An increase in the activity between the start of 25 the euiture period and any later time point is an indication that SSCs have been proliferated. Methods of measuring the activity of SSCs are described in detail eisewhere herein. SSCs that can be proliferated aecordmg to the present invention include, but are not limited to, bovine SSC, human SSC, mouse SSC and rat SSC,

AdditiOnaily, the degree of proliferation of SSCs in a feeder-free euiture system of 30 the present mveniion may be assessed by counting the :aumber of cells: present at a specific point in time during SSC ceU culture and comparing the value to the number of cells present at the start of the culture period . Based: on the disclosure .set forth herein, the

Ο (N

(D GO ski 11 ed artisan wil 1 understan d ih at these and oih er TTiethdds of assessin g SS G rn ai ntenan ee and proliferation may be used. These methods inGludej but itre not limited to, FAGS and MACS, O in (N (N in

o (N 20 25 30

Traiisplantation of Spermatogonia! Stem Cells In one aspect of the inyention, One Or more SiSGs can be transpl anted in to a recipient testis, Transplantaiion methods are generally Imo-vvii in the art, and will not be discussed in extensive detail herein. For general cell transplantation methods involving the testis, see Kanatsu-Shinohara et al, (PNAS, 99:1383-1.388 pdG2)), Brinster (1) (G,S, Pat. No. 6,215,039), and Brinster 01) (U.S. Pat,No. 5,858,35¾ all of which arete incorporated by reference herein in their entirety; Brinster (1) and (II) demonstrate, in part, that SSCs imnsplanted from a donor to an immimologicaily tolerant monse or other compatible reeipient will repKeate and be maintained in the recipient. The feeder free sj«tem is a great advance over traditional feeder systems in that for these to be transplanted the feeder cells bave to be removed. Applicants’ culture, being feedermelTfree, does not require this laborious and difficult step. In an embodiment of the invention, one or more SSGs are introdueed into the tubules of a testis. For example, a recipient mate mammal can be anesthetized and the testis (or testes) surgically exposed. In one erabodiment, using micromanipulation methods, a thin glass needle is introduced into exposed tubules, one after another, and each tubule vs injecied with a solution coniaining the primitive eeils being used to colonize the tubule. In another embodiment, one or more SSGs can also be introduced by infecting them into other parts of the tubular system, e.g. the lumen of the rete testes. As will be understOOd by the skilled artisan, injection methods are available that rninimize the number of injection sites and inereaSe the efficiency of injection of SSGs into a reeipient male. A cell suspension of one or more SSGs for injection can comprise aninj ection medium and at least one SSG at a suitable eoncentration. By way of a non^limiting example, the injection medium can comprise one or more of NaCl, Νβ2ΒΡΌ4, KGl, KH2PO4, EDTA, pyruvate, laeiate, glutamine, glucose;, bovine serum albumin, and DNAse: I. The pH of the iryection media is suitably in the range of 7;0^7,2, but as will be: understood by the skilled artisan, can be acyiisted to be more basic or more acidic 19 ΙΟ

ο (Ν α (υ GO depending upOB the medium Gornpositicjn, the cell tj?pe and/6f concentration, and the microenvironment of the recipient injeetiQn site. ο ΙΟ (Ν (Ν ΙΟ Ο (Ν

In another ernbodiment of the invention, other systems can be used for the introduction of one or more SSGs into a recipient male. These include injection into the 5 vas deferens and epididymis or manipulations on fetal or juvenile tesies, teehniques to sever the seminiferous tubules inside the testicular covering, 'Au th minimal trauma, vvbicb allotv injected cells to enter the cut ends of the tiibules. Alternatively neonatal testis (or testes), which are still undergoing development, can be used.

As set forth elsewhere herein, SSCs entering the testicular tubule are generally 10 protected from destruction by the immunoiogicaily privileged environment of the internal lumen of the tubule. Cells that leak from the tubule are typically destroyed by the immune •system of the host since the cells are foreign to the animal.

In another embodiment of the invention, animal strains are used which are from different speCies to provide donor ceils (xenogeneic transfer). Sources of SSCs include, IS but are not limited to human, rodent, including rat and mouse, primate, including baboon, cow and dog.

The present invention is applicable to any species of animals, ineiuding humans, in which the male has testes, includ ing but not limited to non-human transgenic animals. The invention is also not limited to mammalian species. It can be used toprovide animals and 20 animal lines of many types with a single, or many, novel genetie modifiGation(s) or novel characieristic(s). The animals to wmich the present invention can be applied include humans, non-human primates (eg., monkeys, baboons), laboratory animals, such as rodents (eg., mice, rats, etc.), companion aniruals (eg,, dop, eats), birds (such as chickens and turkeys), wild animals (eg., buffalo, wolves), endangered animals (eg., elephants, leopards);, 25 and zoo species (such as tigers, zebras, lions, pandas, giraffes, polar bears, monkeys, sea otters, etc.) which can be modified to permit their use in cellular diagnosis or assays. The present invention may also be advantageously applied to farm animals, ineluding domesticaied ruminants and fowl (e.g,, cattle, chickens, turkeys, horses, swine, etc.), to imfoue diese animals wifo advantageous genetiG fflodifi.cation(s) or characteristic(s), 30 In another effibpdiment of the invention, the donor and recipient mammal can be the same mammal In one aspect, a popuIatiGn of cells comprising SSCs are coileeied from a mammal prior to destruction of the germ cell population and then reiniroduced thereafter. 20 T|is embodirrieni would presen'e the ability of die marnmai to reproduce following radiation therapy, for example which may be necessary during the treatment of cancer. Alternatively, spermatogonial stem cells may be haiwested from the mammal and kept in culture or frozen. lit this aspect of the invention, when progeny are desired, the stem cells 5 are transplanted to a recipient testis. The donor laamxnal egg can then be fertilized by spertnatozoa developed in the recipient testis. There are no time constraints on this procedure since the stem cells continually undergo selTrenewal. ο (N Ph <D in

o (N (N O (N

Methods of fertilization of eggs and progeny are kitown in the art, and will not be discussed in detail herein. By way of a nOn-iiiniting: exampie,, methods of fertil izing eggs 10 include, but are not limited to, intracMopla.smj.c spelTii injectibn (ICSI), round spermatid iniection (ROSi), and the like. Additionally, by way of a non-l imiting example, methods of fertilizing progeny include, but are not limited to j iCST R0Si, and the like.

Once an initial fertilizatiGii event is achieved and the resulting offspring is fertile, the rnaminal line with its novel genetic modification or charaGteristiG is established where 15 the novel genetic modification or charaeteristic is present in both male and: female offspring. Thus, in accordance with the invention, a mammal may be produced harboring, in its testes only, a biologically functional germ cel! which is not native to that mammal by repopuiating its testicular seminiferous tubules. This tpamni); mammal can produce progeny. Every cell in the progeny in genetically nonmative as compared to the parent 20 mammal.

Both the parent mammal·and its progeny provided by^ the pre.sent invention have multiple and varied uses, including, but not limited to, uses in agriculture and biomedicine, including human gene therapy. An illustrative agricultural use of the present invention relates to increasing the breeding potential of a valuable stud anima}. in another aspeet of 25 the present invention, chimeric animals useful in either biGmedicine or agricultuic are provided. As will be understood by the skilled artisan, wbeh armed with the present application, the present inveinion provides an advantageous eomplementaiion to existing transgenic animal tecltnitues.

The present ittvehtion alieviates the present difiiGulty and expense of embryplogical 30 transgenic work . In one embodiment of the present invention^ spermatogonial stem eells can be genetically modified and then transferred to recipient testes. The valuable genetic traits present in the resuhant germ cells can be passed emto the progeny of the 21 recipient stud. This paiticular applicaticin of the present invention is important for the; geneti c engineering of large agricultLiral animals .

ο (Ν ω 0Q ο (Ν (Ν Ο (Ν

As set forth herein, ths pt®sent invention also has appHcations in gene therapy, including human gene therapy. By way of a non-lirntting example, a patient with a 5 deleterious genetic trait could undergo a testicular biopsy. Stem: eells can he geneticaily tnoiiiired to correct th© deleterious trait. The patient then undergoes a tioatment the remaining germ ceils from his testes, for example by speeiflc irfadiation of the testes. His testes (now devoid of germ cells) can then be recolonized by his own, genetically-coitecied, stem cells. The patient can then father progeny free from the worry that he 10 would pass on a genetic disease to his progeny. Alternatively, the stem cells with the corrected gene can be transplanted to a mouse and the resulting sperm used for fertilizing eggs, thereby foregoing the need for reimplanting stem Cells into the original human testis.

Tn another embodiment, the present invention also has applications in establishirig, restori ng or otherwise enhancing fertility in a male mammal, including, but hot limited to, Ϊ5 humans. By way of a non-limiting example, a patient haviiig a disease or disorder treatable by radioisotOpe therapy, chemotherapy, or both, is a donor of one or more SSC. After the patient has undergone radioisotope therapy, chemotherapy, or both, the patient may possibly be rendered devoid of SSC, or maybe otherwise rendered infertile. By using, prep aring or transplanting one or more of the patient's SSC according to tire present 20 invention, fertility in the patient may be established, restored, or Otherwise enhanced.

Sterile Testes Compleinentation

Currently, specific causes of infertility in men remain a mystery in 40-60%. of cases. See Bhasin et al, J Clin Endocrinol Metab 79,1525-9 (1994); Sadeghi-Nejad et al., Urol j 25 4, 192-206 (2007); and Matzuk et al, Nat Med M, 1197-213 (2008). In total, >5% of the male population is infertile, and >1% of all rriales are inflicted with a severe defect in Sperm production termed azoospermia. See Bhasin et al., J Ciin Endocrinol Metab 79, 1S2S‘9 (1994); Sadeghi-N^adet ai,, Urol j 4,192-20:6 (2007); Barthold et al. Jtlrol 1:70, 2396-401 (2003); and Bieyer, W. A. CA Cancer J Clin 40, :355-67 (1990), Fundamentally, 3Q because azoospetmia results in an inability to reproduce by natural mating, it seems enigmatic :as to why this disease remains so prevalent in the human population, :Sueh an epidemiGlogical trend clearly points to the existence of potent ehvironinental factors ftat

Ο (Ν Οη (D GO Ο (Ν (Ν Ο (Ν disrupt the process of sperfti production (i .e. speiTnatogeuesis) or a suhstantial number of de novo mutations that could arise during a lifetirne to render one sterile, but otherwise healthy. See Bhasin et aL, J Clin Endocrinol Metab 79,1525-9 (1994¾ Bleyer, W. A,, CA Cancer J Glm 40, 355-67 (1990); Reyo, R. et al. Nat Genet 10, 383^93 (1995); Oates et ah, Hum Reprod 17,2813-24 (2002). In feet, this is tiue in iium^^ as such de novo mutations acGount for several types of male-factar infertility already defined at a genetic level: ahd increasing numbers of males are left inferiile during their childhood by cancer chemotherapy. See Sadeghi-Nejadj et al., U.rol ,14, 192-206 (2007); Reijo et al. Nat Cenet, 10,: 383-93 (1995); Bleyer et al., CA Cancer J Glin 40,355-67 (1990); Oates et al,, Mum Reprod 17, 2813-24 (2002); Bhasin, S., J Clin Endocrinol Metab 92,1995-2004 (2007); and Geens:, M, et aL, Hum Reprod Update 14, 121-30 (2008). As a new hope for many infertile men with azoospenma, a pioneering breakthrough in stem cell biology that rnanifesied Strong links between reproductive biology and genetic research was the diseovery that mouse testes contained spermatogonial stem cells capable of generating 15 fillly funetiGfial speiTn following isolation and transplantation into testes of another mouse. See Brinster Zimmemiann, Proc Natl Acad Sci USA 91,11298-302 (1994). Similar experiments soon followed in rats, and isolated mouse spermatogonia were next 8110%¾ to maintain their regenerative potential after months in culture. See Clouthier et al , ΝΝίυΓε 381,418-21 (1996); :Hagano et al., Tissue Cell 30, 389-97 (1998). Nevy culture media fiUpporiing the long term proliferation of rodent spermatogonial lines in vi tro have since been fiirmulated and scientists are now on the brinlc of establishing conditions required to cultivate human spermatogonial lines from testis biopsies. See Kahatsu-Shinohara ct al„ Biol Reprod 69, 612-6 (2003); Hamra, F, K. et al, Proe Natl Acad Set USA 102, 17430-5 (2005); Conrad, S. et al. Nature (2008); and Ko-ssaek, N. et al "Isolation and Characterization of Pluripotent Human Spermatogonial Stem Geil-Derived Cells." Stem Cells (2008). Ostensibly, the ability to propagate spermatogonial lines in culture, prior to using them to produce fimctioiial spermatozoa by transplanting them back into the testes of their own donor, presents a clear strategy to cure many existirig types of male infertility. Due in large part to the multipotent nature of germline stem cells however, before ibese breakthroughs are translated into practice it is imperative that pfeeHnical details of such eeilular therapies first be stringently evaluated in more advauGed,:n0n-hriman recipients of medical reievance. See Geens, M. et al, Hum Reprod Update 14, 121-30 (2008¾. Conrad, ίο 20 30 .23 S, et al "Generation of pluripotent stem cells from adult liBman testis," Nature (2008); Kossack, N. et al. "Isolation and Characterization of PIuripotent Hittaaii Spermatogonial Stem Gell-Derived Cells.” Stem Cells (2008); Hermaiin» B. p, et ai. Stem Cells 25, 2330-8 ); and Zhang et al, J Cell Physiol 211,149-58 (2(

ο (N α ω GO

Kits

O (N (N in O (N 3^he invention: includes various kits which comprise a: feeder fee culture system ibr the maintenance or pmliferation of at least one SSC, Although exemplary kits are described below, the contents of other useful kits will be apparent to the skilled artisan in 10 light of the present disclosure. Each of these kits is ineluded within the invention.

In one aspect, the invention features a kit ipr maintaining at least one SSC in a feeder-free culture system, eOmprising a culture system comprising: serum-free defined culture medium and pre-conditioning feeder cells from one or more proprietary cell lines ineluding cell in BEFi and cell line BSCi, an applicator, and insinictional rnaterial, 15 wherein the instructional rnaterial comprises instructions for the use of the kit to maintain at least one SSC in the feeder-free cultm-e system. In another aspect, the invention features a kit for proliferating at least one SSC in a feeder-fee cuiture system, comprising a culture System' comprising serum-free defined culture medium: arid preconditioning feeder cells, or media that has already been pre-conditioned, an appheator, and instruciional rnaterial, 2Q wherein the instructionai material comprise.s mstructions for the use of the kit to proliferate at least one SSC in the feeder-fee culture system.

The invention also features a kit for administering a population of enriched SSCs to a mammal, comprising a culture system comprising feeder-free defined culture medium preconditioned with feeder cells, an applieator, and instmetional material, wherein the 25 instructional material includes instmetions for the use of the kit to proliferate at least one SSC in the feeder-fee culture system and for the applicator-based administration of the enriched SSCs to a mammal.

The partieular appheator included in the kit wilt depend on, e,g., tfe method ahd/or the eomposifion used to introduce a population of enriched SSCs to a cell. Such 30 appiiGatQrs are weh-knGwm in the art and may include, among other things, a membrane, an implant, a syringe, and the Kke. Moreover j the kit Comprises an instruci iona 1 material for the use of the kit. These instructions simply embody the disclosure provided herein.

M

The kit may also include a phanroaeeuiicaliyTaGceptable earner. The composition is provided in an appropriate amount as set forth elsevsfheie herein. Further, the route of administration includes, but should not be limited to, direct contact mth the desired site of adroinistration, as well as contact with a cell or tissue adjacent to the desired site of administration. in ο (N pH in

o in (N (N in o (N

Compositions and methods for the isolation, pimfieation, enrichment, proliferation and maintenance of SSG, as encompassed by the kits of the mveniion, are described in detail elsewhere herein. !0 20

The invention is fiirther described in detail by reference to the following expenmental examples. These examples are piOvided for purposes of illustration only, and are not intended to be limiting unless otherwise speeified. Thus, the invention should in no way be construed as being limited to the following exarapies, but rather, should be constmed to eneompass any and all variations which become evident as a result of the teaching pro\dded herein,

EXAMPLES

Isolation and Culture of Bovine or Porcine Undifferentiated Spemsatogonia 1, Prepare collagenase/DNase incubation solution (l-5mg,''ml coliagenase, Worthington Biologicai and 5-lOmg/mi DNase, Sigma-^Aldrich), 2. Manually tease apart l 00-200mg of dissected bovine or porqme testiGiilar parenchyma using micro forceps and place into collagenase/DNase digestion solutior!. 25 3> InGiibate at 37°C with gentle swirling at a periodic interval uhtil seminiferous tubules have separated. 30 4, Aliow'^ semini ferous tub ule fragments to settle on ice, remove supernatant and wash in Hanks Balanced Sait Solution avmiable from lim Teidmologies Inc;,, Carlsbad, CA 92008 USA, (HBSS) or suitable physiologieal buffer. Repeat procedureunitl supernatant is relatively clear. 25 6. After last settling ineubaiion, remove supematam anci re-:suspend tebb!^ in trypsin/DNase digestion buffer (Invitrogen). Ο (N <ϋ 7. InGubate at 37°C and then add more DNase soiistion fbUowed by pipetting to make a 5 single eell suspension consisting of germ cells and Sertoli cells.

O (N (N o (N 8, Add fetal bovine serum or other Suitable protein source to stop tr>psin digestion and: pass the cell suspension through a suitable ceil strainer to remove tissue fragments thereby yielding a single cell suspension, id 9. Centrifrjge at 6G0xg for 7 min at 4°G to pellet cells. 10. Aspirate supernatant and re-.suspend ceils in DPBS-S (Pliospbate buffered saline with 1% FBS, l OinM Hepes, ImM Pyruvate, Img/ml glucose, 1x10"’u/ml penicillin,, ΙχΙΟ"* 15 pg/ml Streptomyein) or other suitable physiological buffer. 11. Overlay cell suspension onto 30% percoll solution at a density of 2mi percoll solution per 5mi cell .suspension. Other volurnes and ratiospfpereoll and cell suspension may'^ also be suitable. 12, Centrifuge at 6G0xg for 8 min at 4°C. 13. Remove supernatant to recover cell pellet and re~Suspend in DPBS-S or other suitable buffer. 14. Centrifuge at 600xg for 7 min at 4®C. 15, Remove supernatant and re-suspend in StemPiOiCulture medium (recipe below). 30 16. Centrifuge again and wash cells in StethPro to remove residual fetal bovine serum. 17. Re-suspend washed ceil pellei in SteraPro and add gfOxvth factor coektaiI (Cjt>NF 40 ng/ml, FGF 10 ng/mi, CSF-l IQ ng/mi, SDF-1 10 ngdnl) . Other Goncdntrations of these groxvth factors may be suitable.

ο (N α (D GO 5 18. Add cells to 6-vvell culture plates pre-coated with 0.1% gelatin at a density of 2x10-

O uo (N (N uo O (N cells per well and incubate overnight at 37°C. Other culture plate farmats, gelatin Goncenirations, and cel! densitevS may be suitable, 19. Next day, add HBSS or other suitable physiological bufffer to each well and gently ! 0 pipette throughout the well to remove non adherent cells. 20. Collect suspension of non-adheient cells which is eimched for bovine spermatogonia 21. Centriftige the germ cells at 600xg for 7 min at 4°C\ renrove the supematant and rets suspend in B.EF 1 or BSC 1 feeder cell conditioned StemPro media and add growth factor cocktail, 22. Place cells into 24-well plates pre-eoated with Matrigel (BD Bioscienees). Other .substrates such as Laminin and Fibroneedn may also be suitable for coating of plastic 20 culture wells and other culture plate formats including: 96-weli, 48-well, and 12-well may be suitable. 23. Incubate cells at 37°C in an atmosphere of 5% CO2 in air, BEF! or BSCl preconditioned media is changed every other day and cells are re-plated onto new matrix coated plastic plates at an interval of 7-10 days-

The cells can be maintained in these conditions than 6 months and form elumps with morphology as shown in Figure 1. In addition, the cells retain expression of rnolecular makers of undifimentiated spermatogonia including foe transcription fector 30 PLZF. 27

Ο (N α (D

Preconditioning of StemPro Media 1. Maintain BEF andBSG feeders in Duibeced's Modified Eagle Mediuni ilJMiMJ growth media commercially aYailable from Life Technologies, Inc,, Carlsbad, CA 92008 USA and sub-oilture 1:2-1:5 at 80% cOnfluerice.

O (N (N O (N 2. PrecQiidxtiomng of StemPro occurs 24 hr prior to adding the media to cultured Speraiatogonia, 3. Remove DMEM gi'owth media and wash cells w'ith MBSS or other suitable 10 physiological buffer and discard. 4. Add SieraPro media to feeders and incubate at 37°C Gvemight. S, Collect precondition StemPro media and filter through Q.45pm s>mnge filter, add IS factor cocktail (GDNF,FGF2,CSF-1, and SDF-1). 6, Add to bovine spermatogonia being maintained in piastio culture wells coated with Matrigei or othr suitable matrices.

2Q

Creation of Bovine Embryonie Fibrobiasi (BEF) feeders 1, Collect a 3S day old bovine embryo into PBS. 25 2. Remove head and visceral organs induding the gonads. 3. Pice ernbryo iiito small sections with a sterile razor blade. 4, Pigest the tisisue in trypsin solution by incubation at 37“C, 30 5, Allow' tissue to settle on ice theft pipette vigorously. 6. Add more trypsin solution aud incubate at 37'^C. 28

ο (N <D OO

O in (N (N in o (N 7. Allow tissue to settle oii ice, collect superaataut and eentrifuge at 300xg for 5 aim. 8. Re-^suspend peileted cells in DMEM growth media and place in culture plates. 9. (3i0w ceils at 37°C in an atmosphere of .5% GQ2 in air. 10. immortalize cells by transfecting with a reeornbinant DNA eonstatot for overexpression of telomerase. Ϊ0

Greatlon of Bovine Somatic Cell (BSC) feeders 1. Bovine testis cells that adhere to gelatin coated culture wells during the differential plating procedure lor isDlation of spermatogonia are maintained in DMEM growth media 15 after the spermatogonia have been removed. 2. This eell population is a mixture of somatic cells from the bovine testis including SeriOli cells, Leydig cells, and fibroblasts. 2(1 3>: The cells are growi at 37“C in an atmosphere Of 5% GQa in air and arc immortalized by transfection with a recombinant DNA construct for o verexpression of telomerase.

Ο (N α (D GO

O (N (N O (N

StemPro media for Final cone. Supplier bovine undifferentiated spermatogonia Reagent DMEM/F12 tovitrogen ' Penicillin &amp; P; 50 U/ml S:50 ug/ml Invitrogen Streptomycin- Iron-saturated lOug/'ml Sigma Transferrin Na2S.e03 3X10‘*M Sigma 2-ME 50uM Sigma Insulin 5ug/ml Sigma Putrescine 60uM Sigma -M.EM Non-Essential lx invitrogen Amino Acids MEM Vitamins lx Invitrogen Fetal Bovine Sernra (FBS) 0.1% invitrogen Glntamlne 2mM ..... Invitrogen StemPro Supplement 2% Invitrogen 30

Ο (N α (D GO

O (N (N O (N 10 15 20 25

Deposits A deposit of cell lines BBFl and BSCl a-e and have been maintained by an inventor of the this appliction, since prior to the filing date of this application . Access to this deposit will be available during the pendency of the application to the Commissioner of Patents and Trademarks and person determined by tlie Commissioner to be entitled thereto upon request. Upon allowance of any clamis in the applicationj the Applieant(s) Will make avaiiabie to the piiblie without restriction a deposit of each line with the American Type Culture Collection (ATCC), Manassas, Virginia, 20110. The cell lines deposited witli the ATCC will be talcen from the same deposit maintained by the inventors and described above. Additionally, Applicant(s) will meet all the requirements of 37 C.F.R. §1.801 - 1.809, including providing an indication of the viability of the sample when the deposit is made. This deposit of cell lines BEF l and BSC l will be maintained in the ATCC Depository, which is a public depository, for a period of 30 years, or 5 years aier the most recent request, or for the enforceable life of the patent, wihchever is longer, and W'ili be replaced if it ever becomes nonviable during that period. Applicant will impose no restrictions on the availability of the deposited material from the ATCC; however, Applicant has no authority to Waive any restrictions imposed by law on the transfer of biological material or its transpoiiation in commeree.

Throughout this speeifieaiion, unless the context requires othervrise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or method step or gi'oup of elements or integers or method steps but not the exclusion of any element or integer or method step or group of elemenis or integers or method Steps.

Reference to any prior aft in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in any country. 31

Claims (13)

1. CLAIMS:

   1. A method of enriching spermatogonial stem cells (SSCs) from a population of testis-derived cells containing at least one SSC, said method comprising: a) providing a media that has been preconditioned with a feeder cell line, wherein feeder cell line is from bovine or porcine origin; b) contacting said population of testis-derived cells with said preconditioned media under conditions suitable for SSC cell maintenance and enrichment.
2. 2. The method of Claim 1 wherein preconditioning includes the steps of: a) contacting media with feeder cells selected from the group consisting of bovine or porcine embryonic fibroblast cells and/or testicular somatic cells; b) allowing said cells to reproduce on said media; and thereafter c) removing said feeder cells.
3. 3. The method of Claim 1 wherein said spermatogonial stem cells are bovine cells.
4. 4. The method of Claim 1 wherein said spermatogonial stem cells are porcine cells.
5. 5. The method of Claim 1 wherein said pre-conditioned media is DMEM/F12 media.
6. 6. The method of Claim 1 wherein said pre-conditioned media is supplemented with serum replacement supplement.
7. 7. A pre-conditioned media for maintenance and enrichment of mammalian spermatogonial stem cells created by the method of Claim 2.
8. 8. A population of enriched spermatogonial stem cells created by the method of Claim 1.
9. 9. The population of cells of Claim 8, wherein said cells are bovine or porcine cells.
10. 10. A method of generating at least one non-human mammal progeny, said method comprising: a) administering a population enriched SSCs produced by the method of Claim 1 to a testis of a male non-human recipient mammal wherein feeder cells do not need to be removed from said population; b) allowing said enriched SSCs to generate a colony of spermatogenesis in said nonhuman recipient mammal; and c) mating said non-human recipient mammal with a female non-human mammal of the same species as said recipient non-human mammal.
11. 11. The method of Claim 10, wherein said population of enriched SSCs is administered to the lumen of a seminiferous tubule of said recipient non-human mammal.
12. 12. The method of Claim 10, wherein said recipient non-human mammal is infertile.
13. 13. The method of Claim 10, wherein said recipient mammal is selected from the group consisting of a rodent, a non-human primate, a dog, a cow (or bovine), and a pig.