Identification and characterization of repopulating spermatogonial stem cells from the adult human testis

Izydyar et al., 2011. Copperman Lab. 

- Cells were collected from obstructive azoospermic men with normal morphology that indicates normal in-progress spermatogenesis in these men.
- Spermatogonia appear to be round and high nuclei/cytoplasm ratio.

Flow cytometry:
- 13.3 % of the cells express SSEA-4 on their surface. 
Other spermatogonial markers used in this study were CD49f (alpha-6-integrin ITGA6), CD90 (Thy-1), CD117 (c-Kit) and in combination CD49f+ CD90+ and CD1172 (Triple Stain). 

=> In the adult human testes, 25 % of cells express CD49f and 13 % were CD90 positive; and <1 cells="" font="" found="" human="" in="" nbsp="" of="" population="" stained="" the="" triple="" was="">testes.

Immunohistochemistry:

- Colocalized Thy-1 and VASA in germ cells are only at the luminal part of the tubules, peritubules and interstitial cells, but not in the seminiferous tubules  

- 88.3% of SSEA4 positive cells co-localized with ITGA6 and 100% SSEA4-positive cells were co-localized with VASA, but only 50% ITAG6-positive cells were co-localized with VASA. This indicated that ITAG6 is also expressed at the surface of the testicular somatic cells. Almost all ITGA6 cells do not co-localized with Thy-1. 

- Only 25% of SSEA4-positive cells were co-localized with c-KIT => 2 SSEA4-positive populations in adult testis.

- ITAG6-positiive and c-KIT-positive cells were at the basement membrane of seminiferous tubules.

- LH-r is expressed only in the somatic cells and not germ cells in adult testes.

- After FACS sorting for SSEA4-positive cells, they did immunohistochemical staining for GFRa1 and PLZF. There was a 6.5x enrichment of SSCs positive for GFRa1 and 5.0x enrichment of SSCs positive for PLZF in the SSEA4-positive fraction compared to unsorted cells. 

Real-time PCRs:

- c-Kit, GFRa-1, PLZF, c-RET and GPR-125 were expressed at least 3-fold and up to 7-fold greater in the SSEA4 positive population. Moreover, higher expression level of h-TERT in SSEA-4 sorted cells indicates their high level of telomerase activity and their proliferation capability. Unsorted cells showed only 10% telomerase activity while sorted cells in SSEA4-postive fraction showed 54% telomerase activity compared to hESCs (100%). 

SSC transplantation:

SSEA4 MACS showed an enrichment of 40-50 folds for human SSC population, because 6.8HNP+ cells were found per tubule in mice transplanted with SSEA4 MACS sorted cells while it was only 0.15 HNP+ cells in mice transplanted with unsorted cells. 

Using HNP with SSEA4, ITGA1, and c-KIT, they found that in the population of cells positive for HNP,  14% SSEA4+, 28% ITGA1+, 50% dimly c-KIT+. In addition, only a small population of cells positive for SSEA4 were c-KIT+, while 96% of SSEA4+ cells were ITAG1+. 

43% of HNP+ cells co-localized with GPR-125, indicating that GPR-125 is expressed at the surface of a population of repopulating human SSCs.

20-30% of HNP cells were co-localized with Nanog, showing that 30% HNP cells repopulating mouse testes might have pluripotent characteristics

Co-localization of SSEA-4 with Nanog showed that only some of the SSEA-4 positive cells in the mouse testes express Nanog. 

50% of the SSEA-4+ cells repopulating in the mouse testes co-localized GPR-125 indicating that GPR-125+ SSEA-4+ cells and GRP-125+ SSEA-4- cells are both repopulating spermatogonia.

Percentage of co-localization with HNP after human testis cell transplantation into mouse:

VASA                  100

CD117 (c-kit)      49.8

GPR-125             42.8

LH-R                   0

CD49f                 28.1

CD29                   0

SSEA-4              14.2

Nanog                 28.3

Oct-4                   21

TRA1-60              0

Repopulating SSCs in the adult human testes have phenotypic characteristics of SSEA4+,

ITAG6+, CD90+, GPR-125+ and c-Kit neg/low.

Rodent SSCs are only definitively identified by their ability to produce spermatogenesis when transplanted into the testes of infertile recipient mice

Rodents: 

Although no SSC specific marker in rodents has been identified some markers that are expressed

by stem and/or progenitor cells have been described (e.g. GFRα 1, POU3F1, POU5F1

(OCT4), ZBTB16 (PLZF), NGN3, NANOS2, NANOS3, SOHLH1, SOHLH2, FOXO1,

ITGA6 (α 6-integrin, CD49f), LIN28, ID4, UTF1, CDH1, GPR125, ITGB1 (β 1-integrin,

CD29), EPCAM (CD326), CD9 and THY1 (CD90). 

Mouse spermatogonia:

ITGA6+, ITGB1+ , THY1+, CD9+ , GFRα1+, KIT (cKIT, CD117)−, mitochondrial membrane potential high, Rhodamine 123 (Rho123) low , ITGAV (α5-Integrin, CD51)−, MHC-I−  ALDH (aldehyde dehydrogenase) activity−, and CD45−.

Human:

Spermatogonia on the basement membrane of human seminiferous tubules have the phenotype of SALL4+ , ZBTB16+, UTF1+, UCHL1+, and ENO2+. The majority of cells that express SALL4, ZBTB16, UTF1, UCHL1 and ENO2, do not express the differentiation marker KIT.

Germ cell marker: VASA basement membrane

Stem cell marker: SSEA4 surface marker, basement membrane

                             NANOG, POU5F1, c-Myc, SOX2, KLF4, LIN28

                             

Spermatogonia: ITAG6 (both differentiated and undifferentiated spermatogonia), basement membrane and some of the cells outside of the seminiferous tubules

Differentiated spermatogonia: c-KIT, some in basement membrane and some in luminal compartment of seminiferous tubules. Many contradictory sources studying c-KIT localization.

SSC marker: GPR-125, PLZF, UCH-L1, GFRa1 (receptor for GDNF)

Human cells: HNP

Reconstitution in vitro of the Entire Cycle of the Mouse Female Germ Line

Hikabe et al., 2016. Hayashi Lab.

To reconstitute the oogenesis process, they divided the cultures into three groups
(1) in vitro differentiation (IVDi)
(2) in vitro growth (IVG)
(3) un vitro maturation (IVM)

Female ESC line, BVSCH18 (129+Ter/svj(agouti)xC57BL/6) harboring Blimp1-mVenus (BV) and Stella-ECFP (SC) constructs differentiated to PGCLCs. In IVDi medium, these PGCLCs aggregated with E12.5 female gonadal somatic cells of albino ICR strain to form reconstitute ovaries (rOvaries). The oOvaries was changed to Transwell-COL. And at d4, the medium was replaced with StemPro34-based medium. To prevent multiple oocytes follicle formation , oestrogen inhibitor ICI182780 was added to the culture, so each follicle only possessed one oocyte.  During the IVDi culture, BV (marker for early PGCs) was detected at d3 and appeared to be weaker after 1 week. At 2 weeks, BV disappeared and SC (marker for oocytes and PGCs) were prominent in rOvaries. At 3 weeks, SC-positive primary oocytes were abundant in rOvaries. Cluster of PGCLCs were formed by d5 and follicles were formed around d11 of culture. Foxl2 (marker for grandulosa cells) was present in cells surrounding the oocytes at d21. The IVDi culture yielded a large number of secondary follicle-like structure (2FLs) at 3 weeks. These results demonstrated that a number of primary oocytes were induced from ESCs under these culture conditions.

Oocyte growth accompanied by follicular growth is regulated by gonadotropins. FSH plays a central role in proliferation and maturation of granuolsa cells. IVG medium containing FSH proliferation of granulosa cells layer was limited in 2FLs located at the edge of the rOvaries, indicating that 2Fls in the center portion lack signaling space for cell growth. So individual 2FLs were separated from rOvaries. At 11d, primary oocytes in 2FLs grew to germinal vesicle oocytes. On average, 55 fully grown oocytes were collected from 1 rOvary. When transferring them under the IVM culture, 29% of the germinal vesicle oocytes extruded a 1st polar body. The contamination rate of endogenous oocytes was low. The diameters of SC-positive MII oocytes generated in vitro was comparable to those from in vivo. 78% of the MII oocytes generated in vitro has correct number of chromosomes. These results demonstrated that the MII oocytes were successfully produced from pluripotent stem cells in vitro.

To evaluate oogenesis in vitro, the global transcriptome was analyzed. The data showed that oogenesis in vitro recapitulated the differentiation process in vivo with similar dynamics gene expressions. Repetitive elements were repressed in oocytes and the long terminal repeat (LTR) transposons were upregulated both in vitro and in vivo, demonstrating that the trascripts of transposons enriched in growing oocytes are tightly related to oocyte-specific transcriptional regulation. However, there were some differential gene expression between oogenesis in vitro and in vivo. The DEGs upregulated in MII oocytes in vitro were downregulated from primary oocytes to MII oocytes in vivo. In the other hand, DEGs downregulated in MII oocytes in vitro were upregulated from primary oocytes to MII oocytes in vivo. These results suggested that oocyte growth during IVG and IVM culture was compromised in a subset, or perhaps all, of the oocytes.

To test if the in vitro generated MII oocytes could be used for offspring production, they did in vitro fertilization (IVF) with wild-type sperm of albino ICR strain. They collected viable and colored-eye pups. These pups grew normally without any developmental complications and were fertile in both females and males. Their epigenetics of the imprinted genes were comparable to those from in vivo. These results demonstrated that BVSC18 ESC-derived oocytes were capable of producing viable and fertile offspring. The only down point was that the full-term development of  embryos from in vitro generated oocytes was 3.5% while it was 61.7% using in vivo generated oocytes.

To  verify the success of in vitro oogenesis using this system, they tested 5 other cell lines using the same protocol. The 5 lines included BVSCH14 (another ESC from the same mouse strain BVSCH), 2 iPSC lines from 10-week old tail tip fibroblasts (TTFs), 2 iPSC lines from MEFs.  All of these lines gave rise to fully potent oocytes. These oocytes produced colored-eye pups. These pups grew normally, expressed retroviral genes as expected, and were fertiles in both males and females.

Finally, to demonstrate that these in vitro derived oogenesis could complete the entire cycle of female germ line. They nourished the blastocysts formed by BVSCH18-derived oocytes on embryonic fibroblasts in medium containing CHI99021, PD0325901, and LIF. 72.5% of these blastocysts gave rise to ESCs, called regenerated ESCs (rESCs) to distinguish them from in vivo ESCs. The BCSV reporter genes and  sex chromosomes were segregated to each ESC in the Medelian manner. Then, they extracted these ESCs to while-type blastocysts, they observed that these ESCs contributed to multiple tissues including PGCs of the chimera embryos. The rESC-derived MII oocytes underwent second round of meiosis from the initial ESCs. In conclusion, they successfully found a method to recapulate the entire female germline development in vitro using this system.

[Since they had to use embryonic gonadal somatic cells to form aggregates with the derived in vitro derived PGCs, they can one day substitute these somatic cells with gonadal cell-like generated from pluripotent stem cells.]


[Combined Bisulfite Restriction Analysis (or COBRA) is a molecular biology technique that allows for the sensitive quantification of DNA methylation levels at a specific genomic locus on a DNA sequence in a small sample of genomic DNA.]

Complete Meiosis from Embryonic Stem Cell Derived Germ Cells in Vitro

Zhou et al., 2016. Qiu Zhou Lab.

Specification of PGCLCs from ESCs in vitro:

Germline specification occurs early during embryogenesis, when the primordial germ cells (PGCs) are relocated to the extraembryonic compartment. These germcell progenitors migrate to gonal ridges, undergo proliferation and meiosis to form haploid cells. To demonstrate meiosis occurs in vitro, four criteria need to be satisfied: (1) correct DNA content at individual metotic stages, (2)normal number and organization of chromosomes, (3) appropriate nuclear and chromosomal localization, (4) viable and fertile offspring generated from derived gametes. 

Mouse and human embryonic stem cells (ESCs) and induced  pluripotent stem cells (iPSCs) can undergo epiliblast-like developent program in vitro to induce epiblast-like cells (EpiLCs) that are can specify into PGCC-like cells (PGCLCs), from which they can produce haploid spermatozoa. In this study, they demonstrated a complete in vitro meiosis of murine ESC-derived PGCLCs to generate male spermatid like cells (SLCs) that are capable of producing healthy and fertile offspring via ICSC.

Blimp1-mVenus and Stella-ECFP double transgenic ESC line (BVSC) helped identify PGCs.Stra8-EGFP and Prm1-DsRed double transgenic ESC line (SGPD) identify meiotic spermatids. Both of these ESC lines have normal karyotype and produce completely ESC_derived live offspring that confirm their pluripotency. They first culture the SGPD and BVSC in serum-free and feeder-free conditions with addition of GSK3 inhibitor, MEK inhibitor, and LIF. Then these cells were exposed to ActA and bFGF to induce EpiLCs. Then the induced EpiLCs were continued to be cultured in differentiation medium containing EGF, SCF, LIF, BMP4, BMP8b. In this culture, EpiLCs differentiated into PGCLCs which were confirmed by expression of Blimp1-mVenus and Stella-ECFP after 4-6d. FACS helped isolate 11% of PGC cells from the total population based on PGC markers integrin b3 and SSEA1 (integrin b3+/SSEA1+). In addition, PGC genes were upregulated and somatic cell-related genes were downregulated in these cells, indicating in vitro PGCLC specification. Overall, they demonstrated that BGPD and BVSC ESCs were able to induce into PGCLCs.

Initiation of Meiosis in PGCLCs in vitro in Co-Culture:

Co-culturing PGCLCswith early postnatal testicular cells from KITw/KITw-v mice. It was known that high levels of CYP26B1 in fetal-stage gonadal somatic cells prohibits entry of male germ cells into meiosis by metabolizing endogenous retinoic acid (RA). So, in these early postnatal somatic testicular cells expressed low CYP26B1 levels, which were comparable with those of female fetal gonad at the stage of meiotic induction of PGCs at E13.5. Therefore, post-natal testicular cells of KITw/KITw-v mice provided a suitable environment for meiosis initiation when induced PGCLCs
were cultured with them. Three morphogenes (ActA, BMPs, and RA)-containing medium allowed the expression of Stra8-EGFP after 3 days, confirming the initiation of meiosis of these  PGCLCs. In addition, they also observed the migration of PGCLCs toward Stra8-EGFP-expressing cells to form aggregation colonies within 6d. The Stra8-EGFP-positive colonies were DDX4+ (germ cells), GATA4+ (testis somatic cells), and SOX9+ (Sertoli cells), whereas PGC markers SSEA1 and OCT4 were undetectable. Therefore, PGC/SSC cells had differentiated into SGPD PGCLCs comprising of multiple testis cell types through meiosis. Moreover, BVSC cells expressed downregulated Blimp1-mVenus and Stella-ECFP expression, supporting the hypothesis.

Hormonal stimulation induces the formation of haploid SLCs in vitro:

On d7 of co-culture, they stop providing SGPD with ActA, BMPs, and RA, but instead adding FSh and testosterone (T), and BPE. In the presence of FSH/BPE/T, postmeiotic Prm1-DsRed-expressing cells became apparent on d10. haploid spermatid markers tp1,Prm1, acrosin and haprin were upregualted and 14% of these cells contained half DNA, conforming the haploid spermatid-like cell (SLC) formation. Therefore, all three hormones were required for progression of meiosis.

Chromosome synapsis and recombination in meiosis in vitro:

The meiosis progression was assessed via chromosomal synapsis and recombination which require the initiation and resolution of DNA double-strand breaks (DSBs). This SPO11 and RAD51 foci indicated the presence of DSBs in differentiating cells on d8. In addition, the broad distribution of gamma-H2AX on d8 reflected an association with DSBs and the its disappearance from the autosome region on d10 and accumulation on the unsynapsed sex chromosomes indicated the completion of synapsis. These events recapitulated the pachytene stage spermatocytes in vivo. The detection of SYCP1 and SYCP3 also indicated the progression of synapsis. After d8, 90% of spermatocytes were at the leptotene or zygotene stage of meiosis. On d10, 64% were at pachytene stage, indicating the completion of DSB repair and completion of synapsis. By d12, 50% entered diplotene. These results demonstrated that  meiosis in vitro encompassed synapsis and recombination and was synchronized in the majority of germ cells.  The expression levels of transcription factors during meiosis in these germ cells were correlated with in vivo meiosis. The observed conversion of PGCLCs to SLCs was only 10% (1 PGCLC is supposed to give 4 SLCs). On d12, Dividing Prm1-DsRed-positive cells were detected in co-cultures, indicating the formation of haploid cells.

Healthy fertile offspring produced by ICSC with in vitro derived SLCs:

Sorted SLCs contained a cap-shaped acrosome. Small and round cells were selected for sequencing and most of them 75% were haploid and have normal genome structure. The global transcription profile revealed similarities between in vitro SLCs and in vivo spermatids. ICSC was then used to fertilize oocytes with SGPD-derived SLCs, 80-85% developed into 2-cell stage embryos after activation. ICSC was also fertilize oocytes with BVSC-derived SLCs, 83% developed into 2-cell stage embryos. Bisulfite sequencing indicated a pattern associated with maternal and paternal genetic contributions. Embryos from BVSC-derived SLCs developed into live pups with Blimp1-mVenus and Stella-ECFP transgenes, normal karyotype, and normal weight gain. The proportion of high methylation sites (>80%) of the bVSC-derived pffspirng and control mice were all lower than that of sperm but higher than that of the oocyte. 

"Meiosis

In the first part of meiosis (meiosis I) an unusual type of cell division produces two haploid cells that have chromosomes made up of two sister chromatids. 
The chromosomes pair up (a process called synapsis) to form a structure known as either a bivalent (two chromosomes) or a tetrad (four chromatids). 
Prophase I is the stage of meiosis where the homologous chromosomes pair and exchange DNA (genetic recombination). 
Prophase I comprised of five stages; 1) leptotene, 2) zygotene, 3) pachytene, 4) diplotene and 5) diakinesis. 
    1) Leptotene (Greek for "thin threads"):  chromosomes begin to condense. 
    2) Zygotene (Greek for "paired threads"):  chromosomes become closely paired. 
    3) Pachytene (Greek for "thick threads"): crossing over occurs. 
    4) Diplotene (Greek for "two threads"): homologous chromosomes begin to separate but remain attached by the chiasmata. 
    5) Diakinesis (Greek for "moving through"): chromosomes condense and separate until terminal chiasmata only connect the two chromosomes. 
The chiasmata can be formed anywhere along the chromosome but during diakinesis, the chiasmatic connections are translated to the chromosomes' ends. 
The homologous chromosomes are held together during synapsis by the synaptonemal complex. 
Metaphase I:  Bivalent chromosomes attach to the spindle and align at the metaphase plate. 
The bivalents are randomly oriented with respect to the poles such that chromosomes (maternal or paternal or both) are evenly sorted. 
Only the chiasmata hold the paired homologues together. 
Anaphase I:  Homologous chromosomes resolve the chiasmata and move (as a bivalent) to opposite ends of the cell. 
Telophase I: Chromosomes arrive at the poles and nuclear envelopes form and cytokinesis occurs. 
Meiosis II:  The second part of meiosis (meiosis II) is very similar to mitotic division except that DNA synthesis does not occur between the two stages."

Characterization of Human Spermatogonial Stem Cell Markers in Fetal, Pediatric, and Adult Testicular Tissue

Altman  et al., 2014 (Oct). Nam Tran Lab

This study characterizes germ cell membrane markers of male gonocytes, prespermatogonia, and SSCs in humans via FACS. THY1 and c-KIT are transient markers of gonocytes but not in prespermatogonia and post-natal SSCs. c-KIT is detected in gonocytes and THY1 is detected in somatic compnent in addition to its presence in gonocytes. SSEA4 is only present in gonocytes, prespermatogonia, SSCs, and Sertoli cells from fetal through adult human stage.

Male testis contains two populations of germ cells defined by the relative expression of VASA:
At 13 weeks, VASA dim (VASAD) and VASA bright (VASAB) were present as two germ cell populations. VASAB population represent prespermatogonia, but VASAD represent gonocytes. Both VASAD and VASAB express MAGEA confirming that they are primitive germ cells. In adult testis, VASAD a germ cells were in the basement mmbrane where primitive spermatogonia and SSC are located. In adult testis, VASAB were seen near the lumen where mature spermatocytes are located.

SSEA4 is a common membrane marker for gonocytes, prespermatgonia and Sertoli cells: 
During second and third trimesters, SSEA4 is expressed in all seminiferous cord cells. Both VASAD and VASAB express SEEA4 at similar levels, indicating that SSEA4 is a common marker for both gonocytes and prespermatogonia. SSEA4-positive/VASA-negative are identified as Sertoli cells. In adult testis, SSEA4-positive/VASA-postive were only detected in VASAD germ cells, indicating that SSEA4 remained to be the marker for primitive spermatogonia.

Gonocytes transiently express c-KIT and THY1:
THY1+ cells are arranged in clusters of 3-6 cells and expressed low expression levels of VASAD; however, VASAB never expressed THY1. SSEA4+/THY1+ are considered primitive gonocytes. At 19 weeks of gestation, 10% of SSEA+ cells were THY1+ (gonocytes), and 90% of SSEA4+ cells were THY1- (prespermatogonia and Sertoli cells). In contrast, >90% of SSEA4- cells express THY1.

After second trimester, THY1 expression was detected only in somatic cells outside of seminuferous cords while c-KIT was not detected in either VASAD or VASAB cells. Even though THY1 was found in early fetal gonocytes, THY1 expression in adult men was solely expressed in somatic cells, including Sertoli cells, peritubular interstitial cells, and cells making up the lamina propria.

SSEA4 continues to be the membrane marker or spermatogonial stem cells (SSCs) postnatally:
Both SSC and Sertoli cells from 4 month  and 4 year old boys continued to express SSEA4 within the seminiferous cord, as seen in fetal testis during third trimester. Neither THY1 nor c-KIT expression was detected within the seminiferous cord of these postnatal testis. And less than 10% of spermatogonia were VASAD postnatally.

Reconstitution of the Mouse Germ Cell Specification Pathway in Culture by Pluripotent Stem Cells

Hayashi et al., 2011. Saitou Lab.

In mice, germ cell fate is induced in epiblasts at E6.0 by Bmp4 signaling from extraembryonic ectoderm. The primordial germ cells (PGCs) are established at E7.5 as small cluster of alkaline-phosphate positive cells (AP+) in the extraembryonic mesoderm. Blimp1 and Prdm14 regulate PGC fate. All epiblast cells from E5.5-E6.0 express Blimp1 and Prdm14 in response to Bmp4, than the PGC-like cells derived from epiblasts ex vivo can for functional sperm when transplanted into neonatal testes lacking endogenous germ cells. 

{Summary: germ cell fate: Blimp1 + Prdm4 (in epiblast cells E5.5-E6.0) are upregulated in response to Bmp4 (from extraembryonic ectoderm E6.0) --> PGC cells (in extraembryonic ectoderm E7.5) --> sperm}

Pluripotent cells in mice are comprised of (1) inner cell mass (ICM) of preimplantation blastocyst (E3.5-E4.5) and (2) the epiblast of postimplantation embryos  (E5.5-E6.5). These two populations induce into two pluripotent stem cell populations. The ICM gives rise to embryonic stem cells (ESC) and the epiblast give rise to epiblast stem cells (EpiSCs).

ESCs and EpiSCs show distinct morphology, cytokine dependency, gene expression and epigenetic profiles. ESCs bear  the ground state (naive) pluripotency and can contribute to all linages when introduced into the blastocyst. Whereas, EpiSCs are primed pluripotent and are not able to contribute to chimeras  when introduced into the blastocyst. Interestingly, hESCs are more like mEpiSCs, making it difficult for us to capture the hESCs in nonrodent species. 

There are several attempts to grow sperm in vitro starting with ESCs in both mice and humans based on germ cell markers, but these experiments yielded less than 1% of sperm cells which never produced healthy offspring. In addition, some also demonstrated that EpiSCs can also be potential to generate germ cells in vitro, because its subpopulation expresses Blimp1 under self-renewing process and a minority of them are Stella (Pgc/Dppa3) positive. Stella is a marker for established PGCs. But the PGCs population induced from EpiSCs occurs at low frequency even in the presence of BMP4, and their function in vivo has not been demonstrated. 

Therefore, in this study, they have uncovered the conditions that ESCs and PSC (iPSCs) with naive pluripotency  are induced into pregastrulating epiblast-like cells, which derive into PGC-like cells in mice system to produce viable and fertile offspring. [The reason they did not use EpiSCs to induce PGCs in these experiments because they could not find a suitable conditions to improve previous results in the old studies].

Pregastrulating epiblast-like cells from ESCs: (1)ESCs are cultured in serum-free and feeder-free medium implemented with MAPK inhibitor, GSK inhibitor, and leukemia inhibitor factor LIF to allow these ESCs express a uniform property similar to the ICM/preimplantation epiblast at ground state (E4.5). (2) These ESCs could produce PGCs in a few days after ESCs were introduced into the blastocysts. (3) Continuous presence of Activin A (ActA) and basic fibroblast growth factor (bFGF), ESCs converted into EpiSC-like cells exhibiting similarities to the postimplantation (E6.5) epiblasts!!!!! They reasoned that "ESCs might rapidly differentiate into pregastrulating epiblast-like cells with high competence for the PGC fate under conditions similar to those used to induce EpiSC-like cells."

ESCs were derived from E3.5 blastocysts carrying Blimp1-mVenus and Stella-ECFP (BVSC) transgenes. The higher the %KRS, the higher the maintenance of ESC-like state. Stimulation of ActA and bFGF without KRS resulted in an increased cell death rate. After 2 days cultured in ActA, bFGF, and 1% KRS, even though ESCs are induced into epiblast-like cells (EpiLCs), they quickly die after that, and both ESCs and induced EpiLCs are absent of BVSC expression. Nanog and Blimp1 were down-regulated in EpLCs but continue to express in EpSCs. EpiLCs have properties consistent with pregastrulating epiblasts, but EpiSCs do not.  

PGC-like cell induction from EpiLCs: Next they examined if the EpLCs could be induced into PGC-like cells when culture in conditions that cause induction of epiblast cells into PGCs (GMEM + 15% KRS = GK15, cytokines including BMP4). They induce PGCs using ESCs and d1/2/3 EpiLCs for 2d in GK15 medium added with LIF but no BVSC signal was observed. They then cultured ESCs and d1/2/3 EpiLCs in medium containing BMP4 or BMP4 with LIF. Strong BV induction (40%) was observed in d2 and d3 EpiLCs, but not in ESCs or d1 EpiLCs. d2 EpiLCs are highly competent to express Blimp1 in response to BMP4 and cell growth. 

They next examined if  d2 EpiLCs can produce BVSC+ PGC-like cells when culture for longer periods of time. When cultured in full induction condition (GK15 with BMP4, BMP8b, LIF, stem cell factor [SCF]), epidermal growth factor (EGF), BV+ got improved to 16.9% and BVSC+ was 7.2% on d6. This result demonstrated that BVSC+ PGC-like cells were induced by BMP4 and the maintenance/proliferation of BVSC+ cells was enhanced by LIF and even more robustly by LIF, SCF, BMP8b, and EGF. The BVSC+ cells are AP-positive and remained up to 10d under full induction condition. Then, they compared BVSC induction from d2 EpiLCs wth that from epiblast. The efficiency and dynamics of these two BVSC+ cells from these two cell types were the similar.  These results convinced that d2 EpiLCs bear similar, if not identical, properties to pregastrulating epiblast cells. 

Then they evaluated the gene expression profile of PGC-like cells (PGCLCs) induced from d2 EpiLCs. They found that the gene expression dynamics associated with PGCLC induction  were very similar to those associated with PGC specification. They also noted that on d6, the endogenous stella expression in BV+SC- cells was very similar to that in BVSC+ cells, so BV+SC- cells should be considered established PGCLCs. 

Global transcription profiles of EpiLCs and PGCLCs:
The global transcription profile of PGCLC induction was examined from isolated ESCs, d1/2/3/ EpiLCs, EpiSCs, E7.5 epiblasts, and BVSC+ PGCs at E9.5. Based on PC2 scores, the UHC map of nonamplified samples showed that  EpiLC induction from ESCs was a directional and progressive process. EpiSCs were clustered distantly from the other samples, suggesting their divergence from other cell types. The UHC map of amplified samples showed that d2 EpiLC and PGCLCs clustered most closely with E7.5 epiblast and E9.5 PGCs while EpiSCs were more distantly from other cell types. They concluded that the pathway of PGCLC induction from d2 EpiLCs was parallel to that of E9.5 PGC formation from E5.75 epiblasts. [Moreover, the EpiSC induction from epiblast involves a different pathway]. Overall, they demonstrated that the PGCLC formation from EpiLCs derived from ESCs  is a recapitulation of PGC formation form epiblast.  In addition, EpiSCs have more upregulated genes associated with a variety of organ systems than E5.75 epiblasts and d2 EpiLCs, saying that EpiSCs acquire more developmentally advanced characteristics than E5.75 epiblast and d2 EpiLCs.  

Epigenetic Profiles of the PGCLCs:
They evaluated epigenetic profiles of PGCLCs. In ESC to EpiLC differentiation, H3K9me2 and 5mC levels increased and H3K27me3 decreased; but in EpiLCs to PGCL, H3K9me2 and 5mC levels decreased and H3K27me3 increased. 

They next examined PGLCs for the imprinting states of maternally and paternally  imprinted genes. A global decrease of 5mC with relative maintenance of imprinting in PGCLCs was a characteristic observed in migrating PGCs in vivo. Slow growth and G2 cell cycle arrest are characteristics of migrating PGCs. Therefore, PGCLCs is a reconstitution of PGC formation. 

Spermatogenesis and normal offspring from PGCLCs:
PGCLCs induced after 6d were FACS-sorted for BV+ cells. These cells when being transplanted into seminiferous tubules of W/Wv neonatal mice lacking endogenous germ cells resulted in normal spermatogenesis with thicker tubules that contained spermatozoa with normal morphology. Not sorted PGCLCs when transplanted caused testis to develop teratomas (tumor). The efficiency of colonization of the PGCLCs was similar to that of PGCs in vivo.  [Thin tubules usually contained only Setoli cells]. The fertilized oocytes via ICSC with these spermatozoa derived from PGCLCs. They produce viable and healthy offspring (males and females) with normal placentas and imprinting patterns. 

Identification of surface markers for PGCLCs isolation: 
The surface markers of PGCLCs essential for isolating them from stem cells when there are no transgenic reporters bc these PGCLCs are derived from iPSCs or ESCs. SSEA1-high-and-integrin-b3-high population P1 are 99% BV+, correlating well with BV+ PGCLCs. In contrast,  SSEA1-high-and-integrin-b3-low (P2)  or SSEA1-low-and-integrin-b3-high/low (P3) yielded < 2% of BV+. 

To test if this P1 population from AAG ESCs could contribute to spermatogenesis, they transplanted these cells into seminiferous tubules of W/Wv neonatal mice lacking endogenous germ cells. There were no teratomas in testis and showed normal spermatogenesis with GFP signal by AAG transgenes. When fertilizing oocytes with these spermatozoa via ICSC, they produced healthy, fertile offspring.  Therefore, SSEA1 and integrin-b3 could serve as surface makers to isolate PGCLCs from ESCs without contamination of teratomas and relevant transgenic markers. 

PGCLCs. spermatogenesis, and offspring from iPSCs:
They wanted to explore if germ cell production could be possible using iPSCs. iPCS 20D17, 178B-5, 492B-4 lines were used for the experiments. All three lines express Nanog-EGFP (NG) transgene at ground state and shoed differentiation into EpiLCs with proper morphology and NG downregulation. Upon PGCLCs, the NG level was upregulated as early as d2 and NG+ cells formed clusters around the periphery of the aggregates at day 4/6. 

When using FACS to sort SSEA1 and integrin-b3 at d6, only 20D17 line yield similar sorting pattern with ESC lines, and more than 50% of the population was NG+. Consistent with migrating PGCs, the P1 population contained both NG-high and NG-low cells. The expression gene profile of 20D17 line was comparable with BV+ PGCLCs. They then transplanted the P1 isolated from 20D17 population into seminiferous tubules of W/Wv neonatal mice lacking endogenous germ cells. Normal spermatogenesis was observed and norma, fertile offspring were collected. Therefore, although iPSCs exhibit different induction properties depending on the lines, they can nontheless form PGCLCs with proper function. 

Conclusions:
They induced pregastrulating epiblast-like cells (EpiLCs) from ground state ESCs that were maintain by 2i and LIF under serum free and feeder free condition. EpiLCs induction involves Act1 and bFGF, the same cytokines and 1%KRS that are required for the derivation of EpiCLs. d2 EpiLCs were robustly, but not ESCs, d1/3 EpiLCs, or EpiSCs, induced into the PGCs, so only E5.5-E6.0 serve as an efficient precursor for the PGC fate. EpiLCs can serve as a starting material for the induction of other linages derived from the epiblast. 40% of these induced PGCs acquired Blimp1  at a sufficient level proceed to PGCLCs with appropriate genetic, epigenetic, and cellular properties that are compatible to BV+ PGCs formation from epiblast ex vivo.

The identified of surface markers SSEA1 and integrin-b3 for PGCLCs isolation from iPSCs or ESCs. iPSC 20D17 has the highest capacity for germ line transmission among the three lines they used. It was reasoned that efficiency if germline transmission of iPSCs depends highly on Myc gene, and only 20D17 line out of three was derived from retroviral stable transduction of c-Myc. Spermatogenesis of the PGCLCs derived from the iPSCs depends on the original properties of the iPSC lines.

In vitro Production of Functional Sperm in Cultured Neonatal Mouse Testes

Takya Sato et al., 2011. Takehiko Ogawa Lab.

"Neonatal mouse testes [containing] gonocytes or primitive spermatogonia as germ cells can produce spermatids and sperm in vitro with serum-free culture media."  Why serum free? Spermatogenesis can be maintained in the gas-liquid interphase for up to 2 months. Healthy and reproductively competent offspring are born from microinsemination ICSI using these in-vitro-grown spermatids and sperm even when using using thawed cryopreserved neonatal mouse testis.

Sertoli cells are like feeder cells for spermatogonial stem cells.

In the gas-liquid interphase organ culture method, 1-3 mm testis tissue fragment is placed on a piece of agar half-soaked in medium. Gsg2-GFP and Acr-GFP transgenic mouse lines were used to monitor the expression of GFP specifically only in meiosis and  haploid cells via Gsg2 and Acr promoters. In vitro spermatogenesis was proceeded at 34C in aMEM + 10% FBS. FBS is neccessary/indispensible for spermatogenesis in organ culture, specifically round spermatids and haploid cells were observed. However, FBS might contains repressing factors for Gsg2-GFP and Acr-GFP expression. Therefore, they replaced FBS with KSR, which allowed expression of Gsg2-GFP and Acr-GFP. KRS induced stronger and more prolong GFP signal than did FBS. To ensure GFP signal reflects meiosis, meitotic factors SYCP1 and SYCP3 were tested. SYCP1 is colocalized with Acr-GFP during pachytene stage of spermatocytes, but SYCP1 is only colocalized with outer side of Gsg2-GFP positive cells which are finishing meiosis. In somatic cells in this 2.5 dpp mouse testis tissue, Sertoli cells and myoid cells also expressed androgen receptors (AR), a mediator of testosterone effects and essential for spermatogenesi. This result demonstrate that spermatogenesis proceeded in this organ culture condition.

They found spermatids in 6 out of 7 samples (23-50 days) and sperm in 5 out of 11 samples (27-45 days. The sperm formation (1N) was also tested by flow cytometry gating 1N, 2N and 4N. These sperm  were next tested for fertilization via microinsemination. Round spermatids from 3.5 dpp testis cultured for 23 days was used for round spermatid injection technique. Sperm from 2.5 dpp testis cultured for 42 days were used for intracytoplasmic sperm injection (ICSI). Using 23 oocytes for ROSI, 7 live offspring was produced; using 35 oocytes for ICSI, 5 live offspring were produced. 4 out of 12 carried GFP gene because of heterozygous Gsg2-GFP cultured testis. All of these progeny mice were fertile. Cryoreserved testis tissue (4-25 days) grown in the same in-vitro gas-liquid interphase condition as above, 1 out of 5 samples produced results the same as fresh testis tissue cultured in this condition.

It was demonstrated that FBS does not contain factors that inhibit spermatogenesis. They found that lipid-rich bovine serum albumin AlbuMAX was critical in KRS to allow normal spermatogenesis. Addition of only AlbuMAX instead of KRS yielded the same results as observed when using KRS, and using FBS with AlbuMAX also yielded normal spermatogenesis.

First Semester in PhD

I have completed the first semester in graduate school. Throughout these first months, I constantly felt exhausted and anxious all day long. I always felt lost and not doing enough even though I worked like 12 hours a day.  During my undergraduate years, my jobs were more diverse, including classes, research, volunteer, application, part-time job, but I did not feel it was busier than this one semester. In grad school, there is always so much to do even though my jobs were only to do research and study for classes. This one class covered a wide range of biology research topics. The amount of information was enormous and the class was taught by a large group of professors. This gave me a very difficult time to study and handle the exams well. I failed on the first two exams because I was not active in studying for classes but instead spending too much time on exciting research projects. I never failed this bad, this failure in classwork struck me hard. I felt absolutely down and stupid. I started studying seriously and giving up some lab work and fun time. I got my grade back up to A's. I was so happy and gained back my confidence. In the end, I felt so relieved when I passed this class and I will never have to sit in that class ever again but rather moving on to more focused, interesting classes for my research concentration.

I have learned so much during this half-of-a-year time in grad school for how to be a grad student, a good scientist. In short, becoming a good scientist is at a huge expense of personal time, family time, and outside entertainment. The excitement needs to be constantly found in my heart to keep myself motivated and curious to gain my research knowledge and laboratory skills. There are several things I have learned that have helped me becoming a better researcher. Staying focused and critical is crucial in executing experiments to save time and materials and to gain trust from others. The ability to think carefully on every step to troubleshoot technical problems is gained through time and experience. Taking in educational criticism, reading reliable resources, keeping myself updated on new technology, being open-minded, and having research-unrelated hobbies are healthy characteristics that I need to develop, because they will help me enjoy the learning process during PhD, making this path become more enjoyable.

What has been keeping me motivated throughout this time is the enjoyment of being around professionals from where all kinds of interesting ideas are established and shared. I am always amazed by the creativity and the critical thinking that are going on in these scientists' minds. The advanced technology and wide national and international collaborations gave me a number of opportunities to develop myself into a more successful scientist. Obtaining a PhD became so much more challenging than I have ever imagined. All the elements mentioned above together keep me always inspired to continue my training.