TECHNICAL FIELD OF THE INVENTION

The present invention relates to methods for detecting an uncommitted Leydig progenitor cell in a sample of testis cells, methods for selecting uncommitted Leydig progenitor cells, to isolated Leydig progenitor cells, pharmaceutical compositions and the medical use thereof, in particular for the treatment of hypogonadism. The invention further relates to cell cultures comprising uncommitted Leydig progenitor cells and the use of antibodies for detecting

uncommitted Leydig progenitor cells.

BACKGROUND OF THE INVENTION

Spermatogenesis is a continuous process of sperm production and is highly sensitive to factors such as temperature and local hormonal concentrations (Walker WH,2011). The presence of androgens is known to be indispensable for progression of germ cell differentiation and maintenance of the blood-testis barrier, while it simultaneously affects functions of various other organs

(Pakarainen T, et al.,2005; Griffin DK, et al.,2010;Walker WH,2011). Androgens are produced by Leydig cells (LCs), which reside within the testis interstitium.

LCs occur in two forms which are present in different stages of development (Hardy MP, et al.,1991 ;Benton L, et al.,1995;Lejeune H, et al.,1998;Zirkin BR,2010). Fetal LCs arise from mesenchymal-like progenitors within the mesonephros between testis cords and provide the initial onset of steroidogenesis during testis development. During this time, androgens are required for prenatal development of male gonads (Mendis-Handagama SM, et al.,2001 ;Ge RS, et al.,2005). Adult LCs are terminally differentiated steroidogenic cells. Being highly specialized, they are no longer able to proliferate. The population of mature adult LCs are formed and maintained by differentiation of a small population of stem/progenitor interstitial cells from puberty onwards

(Hardy MP, et al.,1991 ;Davidoff MS, et al.,2004). Data obtained from animal studies suggest a low physiological turnover of adult LCs during adulthood. On the other hand, complete restoration of the adult LC population and regeneration of steroidogenic activity have been demonstrated in a rat model after induced LC depletion by administration of ethane dimethanesulphonate (EDS), known to cause apoptosis of mature adult LCs (Teerds KJ, et al.,1999;Davidoff MS, et al.,2004). The functional properties and dynamic changes in the subpopulation of LC progenitors during tissue regeneration are mainly only known from rodent models, but it is generally accepted that this LC regeneration model also holds for the human testis (Davidoff MS, et al.,2004). Undifferentiated pericyte-like cells coexpressing pericyte/mesenchymal, neuronal and glial

(astrocyte/oligodendrocyte) cell markers, that reside in the testicular interstitium in association with the microvasculature system are considered as possible precursors for steroidogenic adult LC (Davidoff MS, et al.,2009). Recently, different ways to isolate steroidogenic progenitors from rodent testis tissue have been described such as a side population approach using Hoechst 33342 or by selection/expansion of 3" -hydroxysteroid dehydrogenase/A-5-4 isomerase (3 β -HSD)-negative, luteinizing hormone receptor (LHR)- negative and a-type platelet-derived growth factor receptor (PDGFRa,CD140A)- positive cells. Cells isolated with each of these methods possess the ability to differentiate towards steroidogenic cells and can colonize testis interstitium of hypogonadal recipients in the rodent (Lo KC, et al.,2004;Ge RS, et al.,2006). However, these cells have a limited proliferative capacity. Without wishing to be bound by theory, the inventors believe that these cells are not uncommitted Leydig progenitor cells but rather a committed cell population already progressed towards mature steroidogenic LC or even a mixed population with most likely partly already fully differentiated LCs. There remains a need for uncommitted Leydig progenitor cells, which may be used for instance in regenerative medicine.

SUMMARY OF THE INVENTION

The present invention is based on the isolation and identification of a new type of adult LC progenitor subpopulation from human testicular tissue (CD 146+ cells). These CD 146+ adult LC progenitors have the capacity to propagate and were able to differentiate towards steroidogenic cells in vitro, indicating these cells have Leydig stem cell characteristics.

In a first aspect the invention therefore provides a method for detecting an uncommitted Leydig progenitor cell in a sample of testis cells based on the presence of CD 146 on the membrane of said cell.

The invention further provides a method for selecting an uncommitted Leydig progenitor cell in a sample of testis cells comprising detecting uncommitted Leydig progenitor cell using the method of detection of the invention and selecting said uncommitted Leydig progenitor cell.

In a further aspect, the invention provides an isolated testis cell, wherein said cell expresses

CD 146 on the cell membrane, said cell being able to proliferate and differentiate toward cells expressing PDGFRa on their cell surface. Preferably, said cell is surface antigen negative for PDGFRa. In a preferred embodiment, said cell is surface antigen negative for CD34. Preferably, said cell is labeled with an antibody against surface antigen CD 146. It is preferred that said cell is surface antigen positive for HLA ABC.

The invention further provides a cell sample wherein at least 75% of the cells are isolated testis cells according to the invention. Preferably, said cell sample comprises at least 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% the cells are isolated testis cells according to the invention.

The invention further provides a method of producing a progenitor Leydig cell or a Leydig cell, comprising expanding and/or differentiating an isolated testis cell according to the invention. The invention further provides a pharmaceutical composition comprising therapeutically effective amount of isolated cells according to the invention or cells obtainable by the method of producing a progenitor Leydig cell or a Leydig cell according to the invention comprising a suitable carrier or medium.

In a further aspect, the invention provides the isolated testis cell according to the invention, the progenitor Leydig cell or a Leydig cell obtainable by the method of producing a progenitor Leydig cell or a Leydig cell according to the invention, or pharmaceutical composition according to the invention for use in the treatment of a disease, more preferably for use in the treatment of hypogonadism.

The invention further provides a cell culture comprising (a) an isolated cell according to the invention; and (b) a medium capable of supporting the proliferation and/or differentiation of said cells.

In a further aspect, the invention provides the use of an antibody against CD 146 for the staining of a testis cell, preferably for the isolation of an uncommitted Leydig progenitor cell. In a preferred embodiment, said use is for the use in any of the methods of the invention.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the isolation of adult LC progenitors from human testicular tissue.

Figure 1 (A) shows a cell suspension obtained from testis biopsy after 1 st enzymatic digestion step during testicular cell isolation (interstitial cell fraction).

Figures 1B-H show the results of a flow cytometrical assay of primary testicular cell suspension before and after culture.

Figure 1(B) shows expression of CD 146 and (1C) PDGFRa cells within the somatic HLA ABC+/CD34- cell population of testicular cells.

Figure 1(D) shows cells expressing CD 146, PDGFRa (IE) and coexpression of these markers (IF) after propagation of testis cells in vitro. Fluorescence-activated cell sorting for CD146+/CD34- (1G) and PDGFRa+/CD34- (1H) cell subsets and recovered cell quantities (I). Scale bar ΙΟΟμιη (ΙΑ). Figure 2 shows the characterization of the sorted adult human Leydig cell population.

Figure 2A shows the results of the quantitative RT-PCR assay for expression of Leydig cell and MSC-specific markers expressed in the two subpopulations of CD146+/CD34- and

PDGFRa+/CD34- cells.

Figure 2B shows the immunophenotype of the sorted CD146+/CD34- cells.

Figure 3 shows propagation of the subcultured sorted adult LC progenitor subpopulations CD146+/CD34- and PDGFRa+/CD34- cells . Figure 3 A shows a subculture of CD146+/CD34- and Figure 3B shows a subculture of PDGFRa+/CD34- sorted cell subsets.

Figure 3(C) shows the cell number and propagation results after subculture of sorted subpopulations CD146+/CD34- and PDGFRa+/CD34- cells.

Figure 3(D) shows the changes in surface markers CD 146 and PDGFRa expression in

CD146+/CD34- cells during in vitro propagation at passage 1, and passage 3 (Figure 3E), and CD146 and SSEA4 expression at passage 1 (Figure 3F) and passage 3 (Figure 3G). Scale bar 100μιη (Α). Figure 4 shows the differentiation to LC in vitro.

Figure 4 shows the differentiation of adult LC progenitor subpopulations CD146+/CD34- (Figure 4A) and PDGFRa+/CD34- (Figure 4B) cells in vitro. Quantitative RT-PCR for expression of LC differentiation-related markers after differentiation- inducing culture conditions at 37°C and 34°C are shown for CD146+/CD34- (4C) and PDGFRa+/CD34- (4D) cell subsets. Figure 4(E) shows the RT-PCR assay for expression of genes indicating steroidogenesis for both cell fractions differentiated at 37°C or 34°C. Changes in CD 146 and PDGFRa surface expression are shown CD146+/CD34- cell subset in differentiation medium (Figure 4F) and control medium as negative control in Figure 4G. 3P~HSD2 expression is shown for CD146+/CD34- cells in differentiation medium (Figure 4H) and in control medium as negative control (Figure 41). Scale bar 50μιη 4(A,B)

Figure 5 shows a model of LC regeneration in adult human testicular tissue.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "uncommitted Leydig progenitor cell" as used herein refers to a cell which is derived from the testis or its fetal precursor gonads and is able to proliferate and differentiate toward cells expressing PDGFRa and luteinizing hormone receptor (LHR) on their cell surface. These cells usually upregulate the expression of steroidogenic enzymes. Uncommitted Leydig progenitor cells are able to proliferate and differentiate toward mature LC at which time they display PDGFRa expression (as well as LHR) on their cell surface and up regulate the expression of steroidogenic enzymes. Said cells possess a higher proliferative activity and have higher expression levels of nestin than committed Leydig progenitor cells. Moreover, when CD146+/CD34-/HLA ABC+ testicular somatic cells are subjected to an induced LC differentiation assay, these cells started expressing the specific steroidogenic enzyme 3- -hydroxysteroid dehydrogenase 2 (3p~HSD2) as well as luteinizing hormone receptor (LHR), which is typical for mature androgen-producing cells.

The term "testis cell" as used herein refers to a cell derived from testicular tissue, preferably cells from the interstitium of the testis or from male fetal gonads, including the mesonephros. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.

As used herein, the term "therapeutically effective amount" means the amount of the pharmaceutical composition that will elicit the biological or medical response of a subject in need thereof that is being sought by the researcher, veterinarian, medical doctor or other clinician.

Embodiments Method for isolating uncommitted Ley dig progenitor cells

The invention provides a method for detecting uncommitted Leydig progenitor cells comprising steps of in vitro labeling testis cells. The uncommitted Leydig progenitor cells are detected based on the presence of the CD 146 protein on the cell membrane. The presence of CD 146 may be detected using any known method. Preferably, specific antibodies which recognize CD 146 are used. Such antibodies are commercially available, for example from BD Pharmingen , San Jose, CA, USA.

Method for isolating uncommitted Leydig progenitor cells

The invention provides a method for isolating uncommitted Leydig progenitor cells, based on the fact that these are testicular cells which are surface antigen positive for CD 146. Therefore, such cells may be isolated by first detecting them, using the method for detecting uncommitted Leydig progenitor cells of the invention as described above, and subsequently isolating the cells which are detected. Methods of isolating labeled cells are known in the art and may comprise techniques such as flow cytometry and cell sorting as described herein or using magnetic beads.

In all aspects of the invention, testis cells are used as a source for uncommitted Leydig progenitor cells. Testis cells may be obtained using methods known in the art. Preferably, testicular cells suspensions may be derived from frozen-thawed fragments of testicular tissue in accordance with previously described protocol (van Pelt AM, et al.,1996 or Sadri-Ardekani H, et al.,2009). In all aspects of the invention, said testis cells are preferably of mammalian origin, more preferably human.

In preferred embodiments of the invention, said testis cells are pretreated by enriching for relevant cells or by removing irrelevant cells. Preferably, said irrelevant cells include endothelial derived from blood vessels and the cells from the seminiferous epithelium. In a preferred embodiment, germ cells and endothelial cells present in a testicular sample are discriminated from the uncommitted Leydig progenitor cells based on HLA ABC and CD34 labeling. Abundant germ cells being HLAABC negative may be removed by using HLAABC membrane markers. Endothelial cells and fibroblasts may be identified using CD34 membrane detection. Cells which express PDGFRa are not uncommitted Leydig cells and may therefore be identified and, if required, removed. Preferably, an interstitial enriched cell fraction is used, preferably after an enzymatic digestion step.

In another embodiment, primary testicular cells are first propagated and subsequently uncommitted progenitor cells of the invention are selected using the selection method of the invention.

Isolated uncommitted Leydig progenitor cells

The inventors have shown that CD 146+ adult LC progenitors have the capacity to propagate and were able to differentiate towards steroidogenic cells in vitro, indicating these cells have Leydig stem cell characteristics. The presence of steroidogenic cells was confirmed by expression of 3 β- HSD2, CYTP450scc, LHR and RLF also known as insulin-like factor 3 (INSL3), all gene previously described as highly specific marker of mature LC (Ivell R, et al.,1997).

The inventors have shown that CD146+/CD34- cells in the human testis represent the population of uncommitted LC progenitors, while PDGFRa+/CD34- cells resemble the early committed cells that derive from these uncommitted CD146+/CD34- cells. Indeed, the uncommitted stem cells are able to proliferate and differentiate toward mature LC at which time they display PDGFRa expression (as well as LHR) on their cell surface and up regulate the expression of steroidogenic enzymes.

In another aspect, the invention therefore provides an isolated testis cell, wherein said cell is surface antigen positive for CD 146. Preferably, said cell is surface antigen negative for PDGFRa. This allows distinguishing the uncommitted LC stem cell from the committed PDGFRa+ Leydig cell progenitor. Preferably, said cell is surface antigen negative for CD34.

Preferably, the isolated testis cell of the invention is bound to an antibody having specific affinity for surface antigen CD 146, to enable easy detection and/or selection.

In another embodiment, said isolated Leydig progenitor cell is surface antigen positive for HLA ABC. In another embodiment, said isolated Leydig progenitor cell does not express CD34 on the membrane. In another embodiment, said isolated Leydig progenitor cell does not express PDGFRa on the membrane. In a preferred embodiment, said isolated Leydig progenitor cell coexpress at least one, preferably all surface markers selected from the group consisting of CD 105, CD73 and CD90.

Method of producing Leydig cells

In order to expand the isolated cells of the invention further, said isolated cells are propagated under different suitable culture conditions. Suitable culture conditions include for example culture conditions reported to be efficient for propagation of rat stem Leydig cells, or similar conditions with minor adaptations (Ge RS, et al.,2006). Media useful for the culturing said isolated cells are both well-known in the art and commercially available and include synthetic culture media and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM; Dulbecco et al., Virol. 8:396 (1959)) supplemented with 4.5 gm/1 glucose, 20 mm glutamine, and 20% fetal calf serum.

In a preferred embodiment, culturing is performed in DMEM/F12 (Gibco), FBS (Gibco), Dexamethasone (Sigma), and further supplemented with human platelet-derived growth factor β- homodimer /BB (PDGF BB) (Sigma), EGF, bFGF and LIF. In another embodiment, culturing is performed in Stem pro complete medium (Sadri-Ardekani H, et al.,2009) or in a medium routinely used for propagation of bone marrow derived MSCs (Gonzalez R, et al.,2009). A person skilled in the art can vary the culture these conditions while getting similar yield.

In a preferred embodiment, said isolated cells are plated, preferably on plastic culture dishes. Subsequently, the cultured cells are sorted using a FACS sorter. Preferably, the sorting is done at passage 3. In a next step, the sorted subpopulations are expanded, preferably on growth factor non-reduced Matrigel (BD, Biosciences). Passaging is preferably done when confluency is reached. The propagated cells may then be passaged using 0,05% Trypsin/EDTA (Gibco) and replated, preferably in 1 :3 ratio.

Induction of differentiation of uncommitted Leydig progenitors in vitro.

Differentiation of uncommitted Leydig progenitors may suitably be performed using the protocol suggested for differentiation of rat LC progenitors with small modifications (Ge RS, et al.,2006), briefly, DMEM/F12 without phenol red (Gibco), 2%FBS (Gibco), InM Dexamethasone (Sigma), 1 Ong/ul human PDGF BB (Sigma), human recombinant insulin- like growth factor 1 (IGF -I) ting(R&D systems), human luteinizing hormone (LH) human pituitary (Sigma), InM

Triiodothyronine (T3) (Sigma) and Thyroxine (T4) (Sigma) ΙΟηΜ, 1% ITS (Gibco),

l%penicillin/streptomycin (Gibco). Preferably, culture medium is supplemented with a compound selected from PDGF, IGF, LH, T3 and T4. Preferably, the temperature is kept between 34°C and 37°C, resulting in formation of subconfluent monolayers.

When CD 146+ testis cells at passage 0 or 1 are subjected to differentiation for 5 and 7 days (under 37°C and 34°C respectively), expression of 3p~HSD2 may be detected accompanied by expression of other markers related to cell steroidogenic function such as CYTP450scc, RELAXIN- LIKE FACTOR (RLF), also known as INSULIN-LIKE FSCTOR 3 (INSL3) and LUTEINIZING HORMONE RECEPTOR (LHR) (Fig.3 c,e). Flow cytometrical analysis performed with the same samples revealed disappearance of CD 146+ surface expression under differentiation conditions accompanied with the appearance of a PDGFRa+ subpopulation varying between 2-5% of the total cell pool. The 3 ~HSD2 expression may be detected only in samples with PDGFRa+ cells. In some embodiments, differentiation is preferably induced for at least 10, 11 or 12 days, to achieve a high yield. To achieve initiation of differentiation, it is in some embodiments of the method preferable that at least a confluence of 30% is reached, prior to application of differentiation media.

Preferably, a medium composition used to induce differentiation of sorted interstitial progenitors towards steroidogenic cells is adopted from known protocol in differentiation assays (Ge RS, et al.,2006)]. Protocols which are described for inducing differentiation of non-human cells, may be adapted to make these protocols more suitable for human cells, in case the method is performed on human cells. In some preferred embodiments, a culturing medium is used, comprising human growth factors and hormones. A suitable culturing medium comprises DMEM/F12 without phenol red (Gibco), 2%>FBS (Gibco), InM Dexamethasone (Sigma), lOng/ul human PDGF BB (Sigma), human recombinant insulin- like growth factor 1 (IGF-I) (R&D systems), human luteinizing hormone (LH) human pituitary (Sigma), InM Triiodothyronine (T3) (Sigma) and Thyroxine (T4)(Sigma) ΙΟηΜ, 1% ITS (Gibco), 1% penicillin/streptomycin(Gibco) (Ge RS, et al.,2006).

Method of treatment of hypogonadism

Male hypogonadism results from a variety of patho-physiological conditions in which testosterone concentration is diminished below the normal range. The hypogonadic condition is sometimes linked with a number of physiological changes, such as diminished interest in sex, impotence, reduced lean body mass, decreased bone density, lowered mood, and energy levels.

Researchers generally classify hypogonadism into one of three types. Primary

hypogonadism includes the testicular failure due to congenital or acquired anorchia, XYY

Syndrome, XX males, Noonan's Syndrome, gonadal dysgenesis, Leydig cell tumors, maldescended testes, varicocele, Sertoli-Cell-Only Syndrome, cryptorchidism, bilateral torsion, vanishing testis syndrome, orchiectomy, Klinefelter's Syndrome, chemotherapy, toxic damage from alcohol or heavy metals, and general disease (renal failure, liver cirrhosis, diabetes, myotonia dystrophica). Patients with primary hypogonadism show an intact feedback mechanism in that the low serum testosterone concentrations are associated with high FSH and LH concentrations. However, because of testicular or other failures, the high LH concentrations are not effective at stimulating testosterone production.

Secondary hypogonadism involves an idiopathic gonadotropin or LH-releasing hormone deficiency. This type of hypogonadism includes Kallman's Syndrome, Prader-Labhart- Willi's Syndrome, Laurence-Moon-Biedl's Syndrome, pituitary insufficiency/adenomas, Pasqualini's Syndrome, hemochromatosis, hyperprolactinemia, or pituitary-hypothalamic injury from tumors, trauma, radiation, or obesity. Because patients with secondary hypogonadism do not demonstrate an intact feedback pathway, the lower testosterone concentrations are not associated with increased LH or FSH levels. Thus, these men have low testosterone serum levels but have gonadotropins in the normal to low range.

Third, hypogonadism may be age-related. Men experience a slow but continuous decline in average serum testosterone after approximately age 20 to 30 years. Researchers estimate that the decline is about 1 -2% per year. Cross-sectional studies in men have found that the mean testosterone value at age 80 years is approximately 75% of that at age 30 years. Because the serum concentration of SHBG increases as men age, the fall in bioavailable and free testosterone is even greater than the fall in total testosterone. Researchers have estimated that approximately 50%>) of healthy men between the ages of 50 and 70 have levels of bioavailable testosterone that are below the lower normal limit. Moreover, as men age, the circadian rhythm of testosterone concentration is often muted, dampened, or completely lost. The major problem with aging appears to be within the hypothalamic -pituitary unit. For example, researchers have found that with aging, LH levels do not increase despite the low testosterone levels. Regardless of the cause, these untreated testosterone deficiencies in older men may lead to a variety of physiological changes, including sexual dysfunction, decreased libido, loss of muscle mass, decreased bone density, depressed mood, and decreased cognitive function. The net result is geriatric hypogonadism, or what is commonly referred to as "male menopause."

Hypogonadism is a frequently observed side effect of gonadotoxic treatment. The inventors are of the opinion that the use of isolated cells according to the present invention is suitable for medical treatment. Isolation, in vitro propagation and transplantation of the isolated cells of the invention (with or without pre- differentiation step in vitro) could then be used to restorate testis steroidogenic function. This transplantation is a good alternative to life-long androgen replacement therapy and help to overcome consequences of endocrine hypogonadism such as insulin resistance, sexual dysfunction and infertility. In a preferred embodiment of the invention, testis cells from a patient are used as source for the treatment of hypogonadism of said patient. Suitably, a cryopreserved testicular biopsy prior to gonadotoxic treatment is used in the treatment. Upon isolation, propagation and transplantion back to the patient after treatment, the stem cells of germ cell- and somatic cell-lineages can restore the testicular function both in sperm production as well as hormonal control of spermatogenesis.

In some embodiments, it is preferred that treatment is performed using the isolated cells of the invention which are differentiated cells using methods herein described. The advantage of using differentiated cells is that the use of differentiated cells may be safer, for example because differentiated cells are less likely to become neoplastic.

In some embodiments it is preferred that treatment is performed using the isolated cells of the invention which only propagated but not differentiated cells using methods herein described. The advantage of using propagated cells of the invention increases the number of the isolated uncommitted progenitor Leydig cells which will allow the turnover to regenerate Leydig cell loss over a long period of time.

Pharmaceutical compositions

The present invention further contemplates therapeutic compositions useful in the treatment of hypogonadism. A subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) or media and one or more of the isolated cells of the present invention, including cells or tissues derived therefrom, alone or in combination with proliferation factors or lineage-commitment factors, including PDGF and LH as an active ingredient.

The isolated cells of the present invention, including cells or tissues derived therefrom, alone or in combination with proliferation factors or lineage-commitment factors, may be prepared in pharmaceutical compositions, with a suitable carrier and in a therapeutically effective dosis for administration by various means to a patient experiencing hypogonadism.

It is a still further object of the present invention to provide pharmaceutical compositions for use in therapeutic methods which comprise or are based upon the isolated cells of the present invention, along with a pharmaceutically acceptable carrier or media. Also contemplated are pharmaceutical compositions comprising proliferation factors or lineage commitment factors that act on or modulate the isolated cells of the present invention and/or the cells, tissues and organs derived therefrom, along with a pharmaceutically acceptable carrier or media. The pharmaceutical compositions of proliferation factors or lineage commitment factors may further comprise the isolated cells of the present invention, or cells, tissues or organs derived therefrom.

A variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. In a preferred embodiment, injection into the interstitium of the testis is performed. The therapeutic factor-containing compositions are conventionally administered intravenously, as by injection of a unit dose, for example. Average quantities of the isolated cells of the invention may vary and in particular should be based upon the recommendations and prescription of a qualified physician or veterinarian.

The preparation of cellular or tissue-based therapeutic compositions as active ingredients is well understood in the art. Such compositions may be formulated in a pharmaceutically acceptable media. The cells may be in solution or embedded in a matrix.

The preparation of therapeutic compositions with factors, including growth, proliferation or lineage-commitment factors, as active ingredients is well understood in the art. The active therapeutic ingredient is often mixed with excipients or media which are pharmaceutically acceptable and compatible with the active ingredient. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.

A factor can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The above disclosure generally describes the present invention. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.

EXAMPLE SECTION

Abstract:

The production of testosterone by Leydig cells (LC) is known to be a required critical factor for progression of spermatogenesis, maintenance of blood testis barrier as a part of specific testicular tissue microenvironment and affects functions of several other organs. LCs are formed at puberty by proliferation and differentiation of adult LC progenitors that reside within the testicular interstitium. The functional properties of adult LC progenitors and how they respond during regeneration, are mainly known from animal studies. Like in rodents, also in the human, testis- specific undifferentiated pericyte-/ mesenchymal- like cells (MSC) that reside in the interstitial perivascular niche have recently been proposed as possible progenitors for steroidogenic adult LC. Here, we determine the charteristics of several testicular somatic subpopulations in order to identify the LC progenitor subpopulation. Our findings suggest that a subpopulation of platelet-derived growth factor receptor a positive (PDGFRa+; CD140A+) human testicular somatic cells represent a population of low-proliferating committed LC progenitor cells, similar as in rodents. In addition, for the first time we found that cells positive for the mesenchymal marker Melanoma cell adhesion molecule (MCAM/CD146+), but negative for PDGFRa, possessed a higher proliferative activity and have high expression levels of nestin. Moreover, when CD146+/CD34-/HLA ABC+ testicular somatic cells were subjected to an induced LC differentiation assay, this subpopulation started expressing the specific steroidogenic enzyme 3- -hydroxysteroid dehydrogenase 2 (3p-HSD2) as well as luteinizing hormone receptor (LHR) typical for mature androgen-producing cells. Our results suggest for the first time the identification of testicular CD 146+ somatic cells as

uncommitted LC stem cells that can be isolated, and propagated and potentially be used for future cell therapy approaches to restore testis steroidogenic function in case of hypogonadism for instance occurring after high-dose chemotherapy.

Introduction

Spermatogenesis is a continuous process of sperm production and is highly sensitive to factors such as temperature and local hormonal concentrations (Walker WH,2011). The presence of androgens is known to be indispensable for progression of germ cell differentiation and maintenance of the blood-testis barrier, while it simultaneously affects functions of various other organs (Pakarainen T, et al.,2005;Griffm DK, et al.,2010; Walker WH,2011). Androgens are produced by Leydig cells (LCs), which reside within the testis interstitium.

LCs occur in two forms which are present in different stages of development (Hardy MP, et al.,1991;Benton L, et al.,1995;Lejeune H, et al.,1998;Zirkin BR,2010). Fetal LCs arise from mesenchymal-like progenitors within the mesonephros between testis cords and provide the initial onset of steroidogenesis during testis development. During this time, androgens are required for prenatal development of male gonads (Mendis-Handagama SM, et al.,2001 ;Ge RS, et al.,2005). Adult LCs are terminally differentiated steroidogenic cells. Being highly specialized, they are no longer able to proliferate. The population of mature adult LCs are formed and maintained by differentiation of a small population of stem/progenitor interstitial cells from puberty onwards (Hardy MP, et al.,1991;Davidoff MS, et al.,2004). Data obtained from animal studies suggest a low physiological turnover of adult LCs during adulthood. On the other hand, complete restoration of the adult LC population and regeneration of steroidogenic activity have been demonstrated in a rat model after induced LC depletion by administration of ethane dimethanesulphonate (EDS), known to cause apoptosis of mature adult LCs (Teerds KJ, et al.,1999;Davidoff MS, et al.,2004). The functional properties and dynamic changes in the subpopulation of LC progenitors during tissue regeneration are mainly only known from rodent models, but it is generally accepted that this LC regeneration model also holds for the human testis (Davidoff MS, et al.,2004). Undifferentiated pericyte-like cells coexpressing pericyte/mesenchymal, neuronal and glial

(astrocyte/oligodendrocyte) cell markers, that reside in the testicular interstitium in association with the microvasculature system are considered as possible precursors for steroidogenic adult LC (Davidoff MS, et al.,2009).

Recently, different ways to isolate steroidogenic progenitors from rodent testis tissue have been described such as a side population approach using Hoechst 33342 or by selection/expansion of 3" -hydroxysteroid dehydrogenase/A-5-4 isomerase (3 β -HSD)-negative, luteinizing hormone receptor (LHR)- negative and a-type platelet-derived growth factor receptor (PDGFRa,CD140A)- positive cells. Cells isolated with each of these methods possess the ability to differentiate towards steroidogenic cells and can colonize testis interstitium of hypogonadal recipients in the rodent (Lo KC, et al.,2004;Ge RS, et al.,2006).

Existing data suggest that it is possible to isolate comparable progenitor cell populations from human testis. Recently, our group demonstrated the existence of bona fide mesenchymal stem cells (MSC), so called multipotent stromal cells, in primary cell cultures derived from human testis tissue (Mizrak SC, et al.,2010;Chikhovskaya JV, et al.,2012). In this study we attempted to isolate adult LC progenitors from this population of testis specific MSCs. We focused on the identification suspensions nor in the cultured human interstitial cell population. While the PDGFRa positive cell show characteristics of committed LC precursors similar as shown for rodents, when using CD 146 as a surface marker we could for the first time identify uncommitted LC stem cells. These LC stem cells might be of interest for regenerative medicine.

Material and Methods

Testicular cell isolation, enrichment and culture Testicular cells suspensions used for isolation of adult LC progenitors were derived from frozen-thawed fragments of testicular tissue in accordance with previously described protocol (van Pelt AM, et al.,1996;Sadri-Ardekani H, et al.,2009), using only the interstitial cell enriched cell fraction recovered after 1st enzymatic digestion step. Tissue specimens of three individuals (URO0059, URO0034 and URO0077) undergoing bilateral orchidectomy for prostate cancer was obtained with informed consent. Histological assessment of the testis tissue sections confirmed the presence of full spermatogenesis.

Isolated cells were propagated under different culture conditions: (I) culture conditions reported to be efficient for propagation of rat stem LC with minor adaptations (Ge RS, et al.,2006), briefly DMEM/F12 (Gibco), 2%FBS (Gibco), InM Dexamethasone (Sigma), lOng/ul human platelet-derived growth factor β- homodimer /BB (PDGF BB) (Sigma), 10 ng/ul human recombinant EGF, 5 ng/ul human, recombinant bFGF, 1 ng/ul human recombinant LIF and 1% ITS, 1% penicillin/streptomycin; (II) Stem pro complete medium (Sadri-Ardekani H, et al.,2009) and (III) medium routinely used for propagation of bone marrow derived MSCs (Gonzalez R, et al.,2009). Primary testicular cells were plated on plastic culture dishes. Cells were sorted at passage 3. The sorted subpopulations were expanded on growth factor non-reduced Matrigel (BD, Biosciences) and after reaching the confluency, the propagated cells were passaged using 0,05% Trypsin/EDTA (Gibco) and replated in 1 :3 ratio. Mesenchymal stem cells as reference

MSCs derived from bone marrow aspirates were kindly provided by Dr. Holger Jahr and applied as a positive control in assessment of expression of specific mesenchymal markers, and cultured in accordance with previously described protocol (Chikhovskaya JV, et al.,2012). Flow Cytometry and Cell Sorting

Flow cytometrical assays were performed on a FACS Canto II flow cytometer (BD

Biosciences) using Diva™ acquisition and analysis software. Primary cultures of testicular interstitial cells at passage 3 were sorted with BD FACS Aria flow cytometer for CD 146+/ CD34- /7AAD- and CD140A+(PDGFRa)/CD34-/7AAD- cells. The following fluorochrome-conjugated antibodies were used for flow cytometrical assays: anti-CD31PE, anti-CD34PECy™7, anti-

CD34FITC, anti-CD44PE, anti-CD45APC, anti-CD49fFITC, anti-CD73PE, anti-CD90APC, anti- CD 105FITC, anti-CD 106APC, anti-CDl 17PE, anti-CD 140A(PDGFRa)PE, anti-CD 146FITC, anti- CD200PE, anti-HLAA,B,CAPC, anti-HLADRAPC, anti-SSEA4 PE (all from BD Pharmingen , San Jose, CA, USA). Anti-CD29FITC was obtained from eBioscience (San Diego, USA) and anti- CD133APC from Miltenyi-Biotec (Bergisch Gladbach, Germany, http://www.miltenybiotec.com). Anti-SSEA4 AlexaFluor 700 labelled antibody was purchased from BD Pharmingen and Invitrogen. Matched isotype controls were applied to determine background fluorescence levels. 7-AAD (BD Pharmingen) was used to exclude non- viable cells from analysis. Due to low concentration of somatic cells in primary testicular cell suspensions 150,000 events were used per single flow cytometrical analysis in order to provide correct interpretation during gating procedure. In vitro differentiation of human adult LC progenitors

In order to assay the differentiation ability of the sorted subpopulations, CD146+/CD34- and PDGFRa+/CD34- cells were propagated up to passages 1-3 before plating under differentiation culture conditions. For each patient enriched subpopulations were tested for their ability to differentiate at 34°C and 37°C in a humidified incubator at 5% C02.

Differentiation of CD146+/CD34- cells was repeated in two independent experiments starting from cryopreserved cells from each patient. Differentiation of PDGFRa+/CD34- cells was performed once per patient, due to low recovery of this cell subpopulation. Sorted subpopulations maintained under propagation culture conditions were used as undifferentiated controls for the differentiation assays. Medium composition used to induce differentiation of sorted interstitial progenitors towards steroidogenic cells was adopted from a previously published protocol with adaptations towards human recombinant growth factors and hormones: briefly DMEM/F12 without phenol red (Gibco), 2%FBS (Gibco), InM Dexamethasone (Sigma), lOng/ul human PDGF BB (Sigma), human recombinant insulin- like growth factor 1 (IGF-I) (R&D systems), human luteinizing hormone (LH) human pituitary (Sigma), InM Triiodothyronine (T3) (Sigma) and Thyroxine (T4)(Sigma) ΙΟηΜ, 1% ITS (Gibco), 1% penicillin/streptomycin(Gibco) (Ge RS, et al.,2006). At 5, 7 and 10 days after the start of differentiation culture, cells were analyzed for expression of differentiation-specific markers by RT-PCR, flow cytometry and

immunocytochemical methods. For each patient enriched subpopulations were tested for their ability to differentiate at 34°C and 37°C maintained in humidified incubator at 5% C02.

Gene Expression Analysis

Isolation of total RNA was performed with the RNeasy mini-kit (Qiagen, Hilden, Germany) accompanied with on-column DNAse treatment by RNAse-Free DNAse (Qiagen,). For reverse transcription, 1 μg of total RNA was used as input with the Superscript II Reverse transcriptase kit (Invitrogen). Conventional PCRs were performed using gene-specific primer pairs (Supplementary data Table SI and SII). In control reactions reverse transcriptase was omitted. The specificity of RT- PCR was confirmed by sequencing the PCR products. Quantitative RT-PCR was performed on a Roche LC 480 instrument using Universal Probe Library assays (Universal ProbeLibrary Assay Design Center www.roche-applied-science.com) (Table SII). Data were normalized for the expression of hypoxanthine phosphoribosyltransferase 1 (HPRT1) as a reference.

Immunocyto- and histochemistry Praffine sections (5μηι) of 4% formalin fixed testicular tissue and 4% formalin fixed cultured cells of interstitial fraction before and after differentiation were stained according to conventional protocol including permeabilization step and omitting the antigen retrieval procedure. Samples were incubated with anti- 3 HSD mouse monoclonal antibody (37-2, SC-10046 Santa Cruz Biotechnology) in dilution 1 :500 at 4°C overnight. Immunocomplexes were visualized with Poly- HRP anti mouse/rabbit/rat reagent in combination with DAB-bright substrate kit (ImmunoLogic). Mouse IgG was applied as a negative control for the immunostaining.

Statistical analysis

Statistical analysis and group comparisons were performed using pairwise fixed reallocation randomization test by Relative Expression Software Tool (REST)(Pfaffl MW, et al.,2002). A value of P < 0.05 was considered statistically significant.

Results

Isolation of two populations of LC progenitors from adult testis

Previously published data supports the concept of perivascular mesenchymal-like cells/pericytes being precursors for adult human LC located within testicular interstitium (Davidoff MS, et al.,2009). The inventors have choosen CD146 (Melanoma cell adhesion molecule (MCAM)) as a surface marker to identify the mesenchymal progenitors. Cell suspensions were obtained from testis biopsies after 1st enzymatic digestion step that are enriched for interstitial cell fractions (ICFs) (Fig. la). While ICF contains augmented amount of somatic cells, HLAABC+ somatic cells still represented only up to 3- 5% of total cells in total fraction (Fig. lb,c). Due to intensive enzymatic disruption of the testicular tissue it is impossible to fully avoid contamination with germ cells. In order to distinguish the target mesenchymal LC progenitors from other cells, colabelling was performed with anti-HLAABC and anti-CD34 to discriminate these cells from germ cells and endothelial cells, respectively. Consequent gating revealed on average 1 7

CD146+/HLAABC+/CD34- cell per 15 000 ICFs (Fig. lb,c).

In accordance with a previously published study reporting enrichment for PDGFRa+ cells as an approach for selection for LC progenitors in rodent model (Ge RS, et al.,2006), we attempted to detect cells with PDGFRa+/HLAABC+ phenotype in ICF suspensions and did not found prominent subpopulation (Fig. lc,). Interestingly, we were also not able to detect cells co-expressing PDGFRa and CD 146 in none of the ICF suspensions, suggesting that two distinct LC progenitor cells may exist in the human testis and PDGFRa-expressing cells may be represented in extremely low frequencies (Fig. 1 b,c). We therefore analysed the stem cell properties of these two separate subpopulations of testicular somatic cells (CD 146+ and PDGFRa+). Propagation potential of CD 146+ and PDGFRa+ progenitors

Taking into consideration that a standard isolation procedure with lcm3 of testicular tissue provides on average 5 xl05 ICF cells, potentially only about 200 30 cells possessing the

HLAABC+/CD146+ phenotypes could be recovered and an even smaller number of

HLAABC+/PDGFRa+ cells. We Therefore we performed initial propagation of ICFs in order to determine their propagation potential and obtain sufficient amounts of HLAABC+/CD146+ and possibly rare HLAABC+/PDGFRa+ cells for further applications. cells for gene expression analyses and in vitro differentiation assays. Three types of culture medium were tested to determine the cell propagation potential in vitro.

All tested conditions provided proliferation of the ICF cells. Flow cytometrical analysis revealed that propagation under culture condition I provided not only proliferation of ICFs but also revealed propagation of the separate CD 146+ and PDGFRa+ somatic cell subpopulations within these IFC population (Fig.ld,e,f). However, expansion under condition II (Stem pro medium) and III (MSC medium) did not allow sufficient propagation of these target subpopulations to facilitate their consequent enrichment by FACS.

The propagated ICFs under culture condition I were subjected to cell sorting to recover the specific subpopulations of CD 146+ and PDGFRa+ cells (Fig. 1 g,h). ICF cell cultures at passage 3 were composed of only somatic (HLA ABC+) cells and possessed substantial differences in CD 146+ and PDGFRa stem cell content between testicular tissues samples from different patients. Interestingly, there was no direct relation between the number of obtained CD 146+ or PDGFRa+ cells and the age of the donor (Fig 2.i), supporting the hypothesis that the stem cell content varies greatly in different individuals.

Obtained sorted CD 146+ and PDGFRa+ cells were subcultured under identical conditions (Fig. 2a,b). During propagation, major differences between these two subpopulations appeared. Whereas CD 146+ cells were able to proliferate and could be propagated after sorting at least up to passage 3, enriched PDGFRa+ cells did not expand in vitro and could be propagated only up to passage 1 (Fig. 2 c). Unfortunately, cell numbers of PDGFRa+ cells at this point were insufficient to perform flow cytometrical analysis. Characterization of CD 146+ and PDGFRa+ progenitors

Obtained sorted CD 146+ and PDGFRa+ cells were subcultured under identical conditions for three passages and subsequently analyzed for the expression of and specific transcripts and surface markers (Fig.2 d-h). CD 146+ and PDGFRa+ cells derived from testicular tissue of three individuals, plated after cell sorting and cultured for 5 days (passage 0) were used to determine the expression of LC-specific markers by quantitative RT-PCR (Fig.2 d). PCR analysis of CD 146- sorted cells revealed beside increased expression of CD 146 transcripts compared to the main cell population, coexpression of high levels of intermediate filament NESTIN and CALBrNDIN 2 (CALB2, CLARETININ) (Fig.2 d). Concerning expression of markers indicating androgen production, this cell population did not express 3 -HSD2 and STEROIDOGENIC ACUTE REGULATORY PROTEIN (StAR), but low level of other steroidogenic enzyme CHOLESTEROL SIDE-CHAIN CLEAVAGE ENZYME (CYTP450scc/CYPl 1 A) could be detected (Fig.2 d). The subpopulation of PDGFRa+ cells did not express 3 -HSD2, NESTIN and CALB 2, although expression of markers indicating steroidogenic activity CYTP450scc and StAR was observed (Fig.2 d).

When successfully propagated CD 146+ cells at different passages were analyzed for the expression of surface markers, a clear decrease in the percentage of CD 146+ progenitors was observed (Fig.2 e,g). At passage 1, a small population of cells expressing the pluripotency marker SSEA4+ existed in the CD 146+ population, which probably remained undetectable during initial FACS analysis due to low numbers of CD 146+ cells (Fig.2 f e). Further analysis indicated that the sorted CD 146+ subpopulation possessed surface antigen expression profile consistent with minimal criteria of MSC identification MSCs (CD29+/CD73+/CD90+/CD105+/CD31-/CD34-/CD45-) (data not shown) (Dominici M, et al.,2006;Gonzalez R, et al.,2009;Chikhovskaya JV, et al.,2012). Further propagation to passage 3 caused subsequent reduction in the percentage of CD 146+ cells (Fig. 2g) and prominent SSEA4-expressing population became no longer detectable (Fig.2h).

Induction of differentiation of CD 146+ and PDGFRa+ progenitors in vitro.

FACS sorted subpopulations of CD 146+ and PDGFRa+ testicular cells were subjected to a differentiation assay according to the protocol suggested for differentiation of rat LC progenitors with small modifications (Ge RS, et al.,2006) Using two different temperatures at 34°C and 37°C.

Due to the limited number of CD 146+ and PDGFRa+ cells at passage 0, we performed from one patient the differentiation assay with CD 146+ cells at passage 0 (i.e. just recovered from FACS procedure) and for the two other patients with CD 146+ sorted cells at passage 1 and 2

(corresponding to 2-3 weeks propagation in vitro). Differentiation assay with PDGFRa+ sorted cells was performed with cells at passage 1 for all three patients.

Throughout the period of differentiation, proliferation of plated CD 146+ cells was observed at 34 DC and 37 DC resulting in formation of subconfluent monolayers. In case of PDGFRa+ cells no proliferation under differentiation culture conditions was observed (Fig. 3a, b.

When CD 146+ cells at passage 0 or 1 were subjected to differentiation for 5 and 7 days (under 37DC and 34DC respectively) expression of 3 ~HSD2 could be detected accompanied by expression of other markers related to cell steroidogenic function such as CYTP450scc, RELAXIN- LIKE FACTOR (RLF), also known as INSULIN-LIKE FSCTOR 3 (INSL3) and LUTEINIZING HORMONE RECEPTOR (LHR) (Fig.3 c,e) Flow cytometrical analysis performed with the same samples revealed disappearance of CD 146+ surface expression under differentiation conditions accompanied with the appearance of PDGFRa+ subpopulation varying between 2-5% of total cell pool. The 3 ~HSD2 expression was detected only in samples with PDGFRa+ cells (Fig. 4c,f,g). Interestingly, differentiated cells possessed significantly higher levels of 3P~HSD2 when cultured at 34° C compared to cells cultured at 37°C (Fig.3a). Generally after 10-12 day, the differentiation assays had to be terminated due to massive cell proliferation. However, overall levels of 3 -HSD expression reduced around this time point at 37°C as well as 37°C, probably due to progressive proliferation of fibroblastic somatic cells overgrowing the differentiating progenitors. In parallel reduction up to 0,1-0,5% in percentage of PDGFRa-expressing cells in relation to total cell pool present in culture was observed. We observed that the initiation of differentiation is highly depended on the cell density prior to application of differentiation media; in wells with low confluence (<30%) differentiation did not take place. Of note, in one of the three patients we were unable to generate steroidogenic cells, most likely because this sample had the lowest recovery of CD 146+ cells and therefore low density at the start of the culture.

When using PDGFRa+ cells in the differentiation assay, quantitative RT-PCR analysis revealed detectable low levels 3 -HSD expression in cultures at all tested time points. However during propagation prior to differentiation, the same low levels of 3 β -HSD expression could be detected, as well as expression of STAR, CYTP450scc and even LHR at some samples.

Furthermore, no increase in 3 -HSD expression during application of differentiation conditions on PDGFRa+ cells has been observed, comparable expression was detected in corresponding negative controls (maintained with propagation medium) at all tested time points . (Fig.3b,d,e ).

Discussion

Apart from the earlier described committed PDGFRa+ Leydig cell progenitors, we described in this study for the first time the isolation and identification of a second types of adult LC progenitor subpopulation from human testicular tissue (CD 146+ cells and cells). These CD 146+ adult LC progenitors have the capacity to propagate and were able to differentiate towards steroidogenic cells in vitro, indicating these cells have Leydig stem cell characteristics. The presence of steroidogenic cells was confirmed by expression of 3 -HSD2, CYTP450scc, LHR and RLF as well known as product of the insulin-like factor 3 (INSL3) gene previously described as highly specific marker of mature LC (Ivell R, et al.,1997) .

Our results supported the hypothesis that mesenchymal, pericyte-like cells harbour an uncommitted progenitor of the LC lineage in adult human testis (Davidoff MS, et al.,2009). The isolated CD 146+ LC progenitors possess CD 146+ expression levels comparable to expression levels of bona fide bone marrow- derived MSCs and coexpress surface markers defining for MSC (CD105, CD73, CD90). The fact that the population of CD146+ cells derived from testicular tissue additionally showed expression of other markers associated with LC progenitor population further supports the possibility to enrich testicular cell suspensions for LC progenitor subset by application of FACS. Sorted CD146+/CD34- cells possess expression of NESTIN intermediate filament protein, previously reported as a marker of human LC progenitors, and CALB2 - calcium-binding protein specific for the LC lineage (Davidoff MS, et al.,1993;Lobo MV, et al.,2004). The resemblance of adult LC progenitors to testicular MSCs is supported by the clinical observation of adipose differentiation and/or areas of ossification within LC tumors caused by transformation of neoplastic LC (Ulbright TM, et al.,2002). This phenomenon suggests that neoplastic LC within testicular tumors, besides features caused by neoplastic transformation, possess the ability to transdifferentiate towards mesodermal lineages. However, the ability to differentiate towards mesodermal lineages is one of the defining universal characteristic of MSCs derived from various organs. The fact that neoplastic LC could spontaneously upregulate these MSC differentiation pathways further suggests close relation between these MSCs and LC precursors. In addition the adipose cells found in these neoplastic LC are usually positive for LC markers (inhibin-a, calbindin 2 and melan-A)(Davidoff MS, et al.,1993;Ulbright TM, et al.,2002).

In contrast to animal studies, our data suggest that PDGFRa+testicular somatic cells do not represent an early uncommitted progenitor, but rather a committed cell population already progressed towards mature steroidogenic LC or even a mixed population with most likely partly already fully differentiated LCs. This hypothesis is supported by the detection of early LC specific markers (CYTP450scc, StAR) in freshly sorted PDGFRa cells and consequent upregulation of 3β- HSD2 expression in these cells during the differentiation assay. The fact that culture conditions that normally trigger differentiation did not affect the appearance of already differentiated cells suggestes that this sorted population is already committed and quite progressed in differentiation towards steroidogenic LC. Moreover, PDGFRa-sorted cells had lower proliferation activity compared to CD 146+ subpopulation that represents the uncommitted progenitors. This observation is in agreement with previous report describing upregulation of PDGFRa surface expression by human embryonic stem cells during differentiation towards steroidogenic cells in vitro (Sonoyama T, et al.,2012).

Based on our results we hypothesize that CD146+/CD34- cells in the human testis represent the population of uncommitted LC progenitors, while PDGFRa+/CD34- cells resemble the early committed cells that derive from these uncommitted CD146+/CD34- cells. Indeed, the uncommitted stem cells are able to proliferate and differentiate toward mature LC at which time they display PDGFRa expression (as well as LHR) on their cell surface and up regulate the expression of steroidogenic enzymes.

Observed consequent transitions in cell surface marker expression profiles corresponding to changes in steroidogenic enzymes expression taking place in vitro, allowed us to suggest the model of adult human LC regeneration. In accordance with our proposal, adult human LC stem cell are represented by CD146+/CD34- MSCs possessing high NESTIN levels located within testicular interstitium. These cells represent the pool of adult stem cells and the source of mature LC turnover in physiological conditions and regeneration in case of acute LC injury. Their transition towards mature LC is going via several intermediate stages/immature LC forms (Fig. 4)

We have to admit that in one out of 3 individuals we were unable to derive LCs in vitro, most likely because this sample provided insufficient numbers of CD 146+ cells and subsequently low density that could not be expanded and differentiated efficiently in vitro. This is most likely due to variability in the retrievable amount of progenitors MSCs between patients. Follow up studies are required to verify the amount progenitors in vivo together with estimation of minimal amount of testicular tissue assuring sufficient derivation of LC progenitors.

The here described CD 146+ LC progenitor population shows stem cell ability in self renewal and differentiation towards steroidogenic cells in vitro. Future studies should establish the ability of the same population to engraft recipient testis and restore androgen production in vivo for instance using the luteinizing hormone receptor knockout mouse model (LuRKO) (Lo KC, et al.,2004;Zhang FP, et al.,2004). The ability to occupy the empty LC niches after xenotransplantation i.e. engraftment into the LuRKO mouse testis would provide essential confirmation on stem cell properties of the derived subpopulation. However, possibility to achieve steroidogenesis in vivo in case of xenotransplantation model appears quite restrained. Further evaluation of stem cell properties may require allotrasplantation studies.

Hypogonadism is a frequently observed side effect of gonadotoxic treatment. Theoretically, isolation of CD 146+ LC progenitors before gonadotoxic treatment would be an appropriate way to preserve the LC pool of patients undergoing gonadotoxic treatment (Chatterjee R, et

al.,2001;Kyriacou C, et al.,2003). Isolation, in vitro propagation and transplantation of enriched autologus LC progenitors (with or without pre- differentiation step in vitro) could then be used to restorate testis steroidogenic function. This transplantation is a good alternative to life-long androgen replacement therapy and help to overcome consequences of endocrine hypogonadism such as insulin resistance, sexual dysfunction and infertility. Cryopreservation of a testicular biopsy prior to gonadotoxic treatment will save the autologus pool of spermatogonial stem cells and LC progenitors. Upon isolation, propagation and transplantion back to the patient after treatment, the stem cells of germ cell- and somatic cell-lineages can restore the testicular function both in sperm production as well as hormonal control of spermatogenesis.

In the present study we identified for the first time the uncommitted Ley dig stem cell. Using the MSC marker CD 146, we could distinguish the uncommitted LC stem cell from the committed PDGFRa+ LC progenitor. The uncommitted Leydig stem cell might be very important in regenerative medicine and further studies on the turnover of LC and regulation of their function. Our findings suggest that isolation of CD146+/CD34 somatic cells from primary cell suspensions obtained after enzymatic digestion of testicular tissue fragments is an appropriate way to derive patient-specific LC progenitors. REFERENCES

1. Benton L, Shan LX, Hardy MP. Differentiation of adult Leydig cells. J Steroid Biochem Mol Biol. 1995:53:61-68.

2. Chatterjee R, Kottaridis PD, McGarrigle HH, Eliahoo J, McKeag N, Mackinnon S, Goldstone AH. Patterns of Leydig cell insufficiency in adult males following bone marrow transplantation for haematological malignancies. Bone Marrow Transplant. 2001 :28:497-502.

3. Chikhovskaya JV, Jonker MJ, Meissner A, Breit TM, Repping S, van Pelt AM. Human testis-derived embryonic stem cell-like cells are not pluripotent, but possess potential of mesenchymal progenitors. Hum Reprod. 2012:27:210-221.

4. Davidoff MS, Schulze W, Middendorff R, Holstein AF. The Leydig cell of the human testis-a new member of the diffuse neuroendocrine system. Cell Tissue Res. 1993:271 :429- 439.

5. Davidoff MS, Middendorff R, Pusch W, Muller D, Wichers S, Holstein AF. Sertoli and Leydig cells of the human testis express neurofilament triplet proteins. Histochem Cell Biol. 1999:111 :173-187.

6. Davidoff MS, Middendorff R, Enikolopov G, Riethmacher D, Holstein AF, Muller D. Progenitor cells of the testosterone-producing Leydig cells revealed. J Cell Biol. 2004: 167:935- 944.

7. Davidoff MS, Middendorff R, Muller D, Holstein AF. The neuroendocrine Leydig cells and their stem cell progenitors, the pericytes. Adv Anat Embryol Cell Biol. 2009:205:1-107.

8. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy.

2006:8:315-317.

9. Ge RS, Dong Q, Sottas CM, Chen H, Zirkin BR, Hardy MP. Gene expression in rat leydig cells during development from the progenitor to adult stage: a cluster analysis. Biol Reprod. 2005:72:1405-1415.

10. Ge RS, Dong Q, Sottas CM, Papadopoulos V, Zirkin BR, Hardy MP. In search of rat stem Leydig cells: identification, isolation, and lineage-specific development. Proc Natl Acad Sci U S A. 2006: 103:2719-2724.

11. Gonzalez R, Griparic L, Vargas V, Burgee K, Santacruz P, Anderson R, Schiewe M, Silva F, Patel A. A putative mesenchymal stem cells population isolated from adult human testes. Biochem Biophys Res Commun. 2009:385:570-575.

12. Griffin DK, Ellis PJ, Dunmore B, Bauer J, Abel MH, Affara NA. Transcriptional profiling of luteinizing hormone receptor- deficient mice before and after testosterone treatment provides insight into the hormonal control of postnatal testicular development and Leydig cell differentiation. Biol Reprod. 2010:82:1139-1150.

13. Hardy MP, Gelber S J, Zhou ZF, Penning TM, Ricigliano JW, Ganj am VK, Nonneman D, Ewing LL. Hormonal control of Leydig cell differentiation. Ann N Y Acad Sci. 1991 :637:152-163.

14. Ivell R, Balvers M, Domagalski R, Ungefroren H, Hunt N, Schulze W. Relaxin-like factor: a highly specific and constitutive new marker for Leydig cells in the human testis. Mol Hum Reprod. 1997:3:459-466.

15. Kyriacou C, Kottaridis PD, Eliahoo J, McKeag N, Bomford J, McGarrigle HH,

Linch DC, Mackinnon S, Chatterjee R. Germ cell damage and Leydig cell insufficiency in recipients of nonmyeloablative transplantation for haematological malignancies. Bone Marrow Transplant. 2003:31 :45-50.

16. Lejeune H, Habert R, Saez JM. Origin, proliferation and differentiation of Leydig cells. J Mol Endocrinol. 1998:20: 1-25.

17. Lo KC, Lei Z, Rao Ch V, Beck J, Lamb DJ. De novo testosterone production in luteinizing hormone receptor knockout mice after transplantation of leydig stem cells.

Endocrinology. 2004 : 145 :4011 -4015.

18. Lobo MV, Arenas MI, Alonso FJ, Gomez G, Bazan E, Paino CL, Fernandez E, Fraile B, Paniagua R, Moyano A, et al. Nestin, a neuroectodermal stem cell marker molecule, is expressed in Leydig cells of the human testis and in some specific cell types from human testicular tumours. Cell Tissue Res. 2004:316:369-376.

19. Mendis-Handagama SM, Ariyaratne HB. Differentiation of the adult Leydig cell population in the postnatal testis. Biol Reprod. 2001 :65:660-671.

20. Mizrak SC, Chikhovskaya JV, Sadri-Ardekani H, van Daalen S, Korver CM,

Hovingh SE, Roepers-Gajadien HL, Raya A, Fluiter K, de Reijke TM, et al. Embryonic stem celllike cells derived from adult human testis. Hum Reprod. 2010:25:158-167.

21. Pakarainen T, Zhang FP, Make la S, Poutanen M, Huhtaniemi I. Testosterone replacement therapy induces spermatogenesis and partially restores fertility in luteinizing hormone receptor knockout mice. Endocrinology. 2005: 146:596-606.

22. Pfaffl MW, Horgan GW, Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res. 2002:30:e36.

23. Sadri-Ardekani H, Mizrak SC, van Daalen SK, Korver CM, Roepers-Gajadien HL, Koruji M, Hovingh S, de Reijke TM, de la Rosette JJ, van der Veen F, et al. Propagation of human spermatogonia! stem cells in vitro. Jama. 2009:302:2127-2134. 24. Sonoyama T, Sone M, Honda K, Taura D, Kojima K, Inuzuka M, Kanamoto N, Tamura N, Nakao K. Differentiation of human embryonic stem cells and human induced pluripotent stem cells into steroid-producing cells. Endocrinology. 2012: 153:4336-4345.

25. Teerds KJ, de Boer-Brouwer M, Dorrington JH, Balvers M, Ivell R. Identification of markers for precursor and leydig cell differentiation in the adult rat testis following ethane dimethyl sulphonate administration. Biol Reprod. 1999:60:1437-1445.

26. Ulbright TM, Srigley JR, Hatzianastassiou DK, Young RH. Leydig cell tumors of the testis with unusual features: adipose differentiation, calcification with ossification, and spindle- shaped tumor cells. Am J Surg Pathol. 2002:26:1424-1433.

27. van Pelt AM, Morena AR, van Dissel-Emiliani FM, Boitani C, Gaemers IC, de Rooij DG, Stefanini M. Isolation of the synchronized A spermatogonia from adult vitamin A- deficient rat testes. Biol Reprod. 1996:55:439-444.

28. Walker WH. Testosterone signaling and the regulation of spermatogenesis.

Spermatogenesis. 2011 : 1 : 116-120.

29. Zhang FP, Pakarainen T, Zhu F, Poutanen M, Huhtaniemi I. Molecular

characterization of postnatal development of testicular steroidogenesis in luteinizing hormone receptor knockout mice. Endocrinology. 2004: 145: 1453-1463.

30. Zirkin BR. Where do adult Leydig cells come from? Biol Reprod. 2010:82:1019-

1020.