Subcutaneous Autografting Leydig Stem Cells: An Approach to Increase Serum Testosterone

Transcription

1 Subcutaneous Autografting Leydig Stem Cells: An Approach to Increase Serum Testosterone Ranjith Ramasamy, MD Department of Urology and Interdisciplinary Stem Cell Institute, University of Miami, FL, USA

2 No disclosures Funding: Research Scholar Award from the SMSNA / AUA Urology Care Foundation

3 Testis performs two important functions Spermatogenesis and Fertility Germ cells Production of testosterone Leydig cells

4 Origin of Stem cell Maturation of Leydig Cell SLC Putative stem Leydig cells Progenitor Leydig cells Immature Leydig cells Up to 11 Days post birth days days Adult Leydig cells By 56 days ALC SLC platelet-derived growth factor receptor (PDGFRa + ) ALC 3b-HSD, LHR Chen, Zirkin et al Li et al. PNAS 2016

5 Testosterone Therapy Side effects Infertility Polycythemia Elevated estrogen Atherosclerosis? MI/ CVA / PE

6 Strategies to increase serum T without affecting hypothalamic-pituitary axis Clomiphene citrate Unlikely to be beneficial in men with elevated LH and testis failure Human chorionic gonadotropin dependent on good intratesticular function

7 Strategies to increase serum T without affecting hypothalamic-pituitary axis Whole testis tissue autograft under skin Makala et al Leydig stem cell transplant Zang et al Major hurdle for clinical translatability

8 Sertoli cells and Peritubular myoid cells are critical for Leydig stem cell survival and function Rebourcet et al 2014

9 Sertoli cells and Peritubular myoid cells are critical for Leydig stem cell survival and function Adjescent CELLS LEYDIG CELLS Adjescent CELLS + LEYDIG CELLS Purified Leydig stem cells Leydig stem cells + peritubular myoid cells + Sertoli cells TESTOESTERONE PRODUCTION IS LOW Not viable TESTOESTERONE PRODUCTION IS HIGH Viable Most probably due to certain Paracrine Factors released by Rebourcet et al 2014

10 Subcutaneous Autografting Leydig Stem Cells: Two Novel Aspects Subcutaneous autograft of Leydig stem cells Autograft of Combination of Leydig Stem cells + Peritubular myoid cells + Sertoli cells US Provisional Patent Application # 62/487,184

11 DIM Day 14 Control Control Relative Expression Relative Expression In Vitro Validation of Leydig Stem Cells from adult WT mice Stem cells are expanded in culture at 14 days Stem cells can Relative be differentiated expression into changes mature upon Leydig DIM cells with LH PDGFRA 3BHSD 3 20 Day 2 Day 2 Day 7 Day DAPI 3BHSD DAPI/3BHSD Control DIM 0 Control DIM DAPI 3BHSD DAPI/3BHSD PDGFRa (+) Negative control

12 Quantification of cells in culture combination of Leydig stem cells with Sertoli cells

13 Leydig stem cells were isolated from testes and autografted into castrate mice

14 Leydig Stem cell were autografted subcutaneously using Matrigel A B C WILD TYPE Orchiectomy Orchiectomy + Autograft After 4 weeks, Animals were sacrificed 1.Serum was harvested Levels were checked a) Testoesterone b) LH c) FSH 2. Skin grafts were obtained 1. Expression were checked a) 3BHSD b) LHR 2. H&E Staining was performed to validate presence of alien cell population in graft

15 Structural Analysis of Subcutaneous Autograft Demonstrated Mature Leydig cells Leydig stem cell injection 10X 40X 60X Recovery of graft at 1 month H&E staining showed the presence of alien cells in the autograft skin

16 Immunofluorescence of Subcutaneous Autograft Confirmed Mature Leydig cells 3BHSD DAPI LHR DAPI/LHR LHR Positive cells LHR

17 Immunostaining of Autograft Confirmed Presence of Peritubular Myoid cells and Sertoli cells and Lack of germ cells a-sma Peritubular myoid cells SOX9 Sertoli cells PLZF Germ cells

18 Testoesterone levels (ng/dl) Testoesterone levels (ng/dl) Leydig Stem cell Autograft Can Increase Serum Testosterone in 4 weeks A A Testoesterone production is detectable B Testoesterone production is Leutinizing detectable B Hormone Leutinizing (LH) production Hormone (LH) production after Stem cell autograft after Stem cell autograft is not affected by autograft is not affected by autograft Neg Ctl Autograft 0 Wild Type Neg Ctl Autograft Wild Type

19 Leydig Stem cell Autograft Can Preserve FSH and LH production A B C D WILD TYPE Orchiectomy Orchiectomy + Autograft Orchiectomy + Testoesterone Pellet 4 WEEKS LATER SACRIFICE ANIMALS, HARVEST SERUM FOR LH & FSH ESTIMATION

20 Leydig stem cells from Human Testis UNDIFFERENTIATED HUMAN STEM LEYDIG CELLS A B Testicular stem cells can proliferate exponentially Differentiated human stem leydig cells (HSLC) A 3β-HSD expression in differentiated human stem leydig cells B A PDGFR-α B 3βHSD

21 Limitations Increase in serum testosterone level is small dosage and duration of autograft needs to be determined Grafts performed in castrate animals to detect changes in testosterone however high levels of LH may have stimulated Leydig stem cell differentiation Levels of LH and FSH remained unaltered by autograft in castrate animals.

22 Summary Leydig Stem cells were harvested, expanded and differentiated in in-vitro conditions both mice and humans. Subcutaneous autograft (Leydig stem + Sertoli + Peritubular myoid cells) were able to maintain a viable population of adult Leydig cells and increase serum testosterone at one month. Levels of LH and FSH remained unaltered by autograft in castrate animals.

23 Future directions Human Leydig stem cell graft in immunocompromised NOD-SCID mice check T, LH, FSH levels Long-term effect of autograft on T, LH and FSH levels ( ~3 months) IND (Clinical trial) A B US Provisional Patent Application # 62/487,184

24 Joshua Hare, MD Dolores Lamb, PhD Lab Members: Acknowledgements Himanshu Arora, PhD Mentors: Tom Masterson, MD