1. Effect of sugar molecules on the cryopreservation of mouse spermatogonial stem cells 
Yong-An Lee, Ph.D., Yong-Hee Kim, B.S., Seung-Jung Ha, B.S., Bang-Jin Kim, M.S., Ki-Jung Kim, M.S., Mi-Seon Jung, B.S., Byung- Gak Kim, Ph.D., Buom-Yong Ryu, Ph.D. 
Fertility and Sterility 
Volume 101, Issue 4, Pages e5 (April 2014)
DOI: /j.fertnstert
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Characterization of PLZF expression by donor germ cells and analysis of effects of sugar molecules on proliferation capacity of thawed germ cells enriched for spermatogonial stem cells. (A) Undifferentiated states of donor germ cells were characterized by immunostaining for PLZF, which is a specific marker for undifferentiated spermatogonia including spermatogonial stem cells. Initial: Immunostaining of testis cells isolated from mouse (C57GFP) pup testis cells before magnetic-activated cell sorting (MACS). Before culture: Immunostaining of Thy-1+ cells isolated from mouse pup testis cells using MACS and anti-Thy-1 microbeads (Before culture; MACS Thy-1+). After culture (top row): Immunostaining of donor germ cells after 6-week culture of MACS Thy-1+ cells. Almost all cells expressing green fluorescent protein (GFP) expressed PLZF after culture for 6 weeks. Arrowhead indicates SIM mouse embryo-derived thioguanine- and ouabain-resistant (STO) feeder cells that do not express PLZF. After culture (bottom row): Immunostaining of the germ cells forming the clumps during mouse serum-free culture revealed that germ cells expressing GFP expressed PLZF. Scale bars = Initial, Before culture, and After culture (top), 75 μm; After culture (bottom), 200 μm. The percentage of germ cells (means ± SEM [n = 3]) expressing PLZF per total cells recovered from the testis cells was 2.5% ± 1.0%, 57.7% ± 1.9%, and 93.8% ± 3.3% before MACS, after MACS Thy-1+ selection, and after 6 weeks of culture of MACS Thy-1+ cells, respectively. DAPI = 6-diamino-2-phenylindole. (B, C) Assessment of putative germ cell function was demonstrated by proliferation capacity, which is determined through germ cell culture after freeze-thawing. Proliferation capacity of germ cells cryopreserved with monosaccharides (B), disaccharides, and trisaccharides (C) was expressed as a percentage normalized to the number of freezing control germ cells (freezing without sugar) recovered after freeze-thaw and culture. White bars = freezing control group; light gray bars = 50 mM sugar molecule group; dark gray bars = 100 mM sugar molecule group; black bars = 200 mM sugar molecule group. Values are means ± SEM (n = 7 independently established cultures for each treatment). Different letters within a sugar group indicate significant difference (P<.05). *Significant (P<.05) difference from 200 mM trehalose treatment group with other 200 mM disaccharide (lactose, maltose, sucrose) treatment groups; n.s. = no significant difference.
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Effect of sugar molecules on stem cell activity after cryopreservation. Assessment of spermatogonial stem cell activity was demonstrated by counting the number of colonies derived from donor spermatogonial stem cells after transplantation. (A) Bright-field image of germ cells in 7 days in vitro culture after thawing from donor cells frozen in the presence of 200 mM lactose. (B) Dark-field fluorescence image of (A). (C) Dark-field fluorescence images of a recipient testis transplanted with germ cells frozen in the presence of 200 mM lactose. Colonies of donor spermatogenesis are distinct green regions of the recipient seminiferous tubules. (D) Cryosections of donor-derived germ cell colonies. Complete spermatogenesis is evidenced by the presence of sperm (white arrow) in the lumen of the seminiferous tubules. (E) The number of colonies per 105 transplanted cells. (F) The number of colonies per total number of cells recovered after freeze, thaw, and culture. Values are means ± SEM (n = 3 experiments per treatment). Total number of mice/testes analyzed were 9/15, 9/18, 8/12, and 12/21 for fresh control: noncryopreserved; freezing control: cryopreserved without sugar molecules; lactose: cryopreserved with 200 mM lactose; and trehalose: cryopreserved with 200 mM trehalose. Different letters indicate significant difference (P<.05) between treatments. Scale bars: (A, B) = 100 μm; (C) = 2 mm; (D) = 40 μm.
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Effects of serum-free condition on stem cell activity after cryopreservation. Assessment of spermatogonial stem cell activity was demonstrated by counting the number of colonies from donor spermatogonial stem cells after transplantation. (A) Bright-field image of germ cells in 7 days in vitro culture after thawing from donor frozen in freezing medium replaced by fetal bovine serum (FBS) with knockout serum replacement (KSR) and supplemented with 200 mM trehalose. (B) Dark-field fluorescence image of (A). (C) Dark-field fluorescence images of a recipient testis transplanted with germ cells frozen in freezing medium replaced by FBS with KSR and containing 200 mM trehalose. (D) Cryosections of donor-derived germ cell colonies. Complete spermatogenesis is evidenced by the presence of sperm (white arrow) in the lumen of the seminiferous tubules. (E) The number of colonies per 105 transplanted cells. (F) The number of colonies per total number of cells recovered after freeze, thaw, and culture. Fresh = noncryopreserved cells (n = 6 samples; 16 total mice and 27 total testes were transplanted); D,S = freezing medium containing dimethyl sulfoxide (DMSO) and FBS (n = 6 samples; 18 total mice and 33 total testes were transplanted); D = freezing medium containing DMSO only without FBS (n = 3 samples; 7 total mice and 11 total testes were transplanted); D,SR = freezing medium containing DMSO and replaced by FBS with KSR (n = 3 samples; 8 total mice and 14 total testes were transplanted); D,S,T = freezing medium containing DMSO, FBS, and trehalose (n = 6 samples; 18 total mice and 32 total testes were transplanted); D,SR,T = freezing medium containing DMSO replaced by FBS with KSR and containing trehalose (n = 3 samples; 9 total mice and 16 total testes were transplanted). Scale bars: (A, B) = 100 μm; (C) = 2 mm; (D) = 50 μm. Values are means ± SEM. Points with different superscripts are significantly different (P<.05).
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

5. Figure 4
Characterization of germ cell marker expression by freeze-thawed germ cells. To compare the undifferentiated state, four groups of germ cells derived from culture of the magnetic-activated cell sorting Thy-1+ mouse pup testis (C57GFP) cells, including noncryopreserved germ cells (fresh control), germ cells cryopreserved in freezing medium containing 10% fetal bovine serum and 10% dimethyl sulfoxide (DMSO) (freezing control medium), germ cells cryopreserved in freezing control medium containing 200 mM trehalose (trehalose treatment), and germ cells cryopreserved in freezing medium containing 200 mM trehalose and replaced by fetal bovine serum with 10% serum replacement (trehalose + SR), were immunostained with antibodies against germ cell markers, including markers for germ cell lineage (VASA), undifferentiated spermatogonia or spermatogonial stem cells (GFRA1 and PLZF), and differentiated spermatogonia (c-Kit). (A–P) Immunostaining of germ cells recovered from trehalose + SR group after freeze-thawing. Scale bars = 75 μm. (Q) The percentage of germ cells expressing VASA, GFRA1, PLZF, and c-Kit in the fresh control, freezing control medium, trehalose treatment, and trehalose + SR groups. No significant difference was observed in the percentage of germ cells expressing VASA (98.7% ± 0.7%, 98.4% ± 0.5%, 98.3% ± 1.1%, and 98.2% ± 0.5% for fresh control, freezing control medium, trehalose treatment, and trehalose + SR groups, respectively), GFRA1 (95.8% ± 0.8%, 95.2% ± 0.8%, 97.5% ± 0.5%, and 96.6% ± 0.5% for fresh control, freezing control medium, trehalose treatment, and trehalose + SR groups, respectively), and PLZF (93.2% ± 1.8%, 95.4% ± 1.1%, 96.3% ± 1.1%, and 97.5% ± 1.3% for fresh control, freezing control medium, trehalose treatment, and trehalose + SR groups, respectively). In addition, germ cells were mostly negative to c-Kit expression (1.7% ± 0.3%, 0.9% ± 0.5%, 1.3% ± 0.6%, and 1.2% ± 0.4% for fresh control, freezing control medium, trehalose treatment, and trehalose + SR groups, respectively), which is expressed in germ cells committed to differentiation. These results revealed that an undifferentiated state is maintained on germ cells recovered after cryopreservation regardless of freezing medium, without difference compared with the fresh group. Values are means ± SEM (n = 2).
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

6. Supplemental Figure 1
Flow cytometric analysis of forward scatter and side scatter patterns for single testis cells from mouse pup testis. (A, B) The forward scatter and side scatter patterns of the single cells isolated from mouse (C57GFP) pup testis cells (testis cells before MACS) and the Thy-1+ cells isolated from mouse pup testis cells using magnetic activated cell sorting (MACS) and anti-Thy-1 microbead (MACS Thy-1+). The major population of MACS Thy-1+ (81.3% ± 3.1%) is contained in (B, gate); a significantly lower percentage (62.1% ± 4%; P<.05) of the testis cells before MACS is held in (A, gate). Values are means ± SEM (n = 2). Cells were stained with propidium iodide (PI) before analysis, and only live cells (PI− cells) were analyzed.
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

7. Supplemental Figure 2
Effects of sugar molecules on recovery rate during cryopreservation of germ cells enriched for spermatogonial stem cells. (A, B) Percentage of viable cells recovered after thawing of the cells frozen with monosaccharides (A), disaccharides, and trisaccharides (B). White bars = freezing control (freezing without sugar) group; light gray bars = 50 mM sugar molecule group; dark gray bars = 100 mM sugar molecule group; black bars = 200 mM sugar molecule group. Values are means ± SEM (n = 4 independently established cultures for each treatment). Different letters within a sugar group indicate significant difference (P<.05).
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

8. Supplemental Figure 3
Comparison of fetal bovine serum (FBS) and knockout serum replacement (KSR) as cryoprotectants for germ cell recovery and proliferation rate. (A) Percentage of viable cells recovered after thawing cells that were frozen with different freezing mediums. There is no difference between the dimethyl sulfoxide (DMSO)-only group (D) and the FBS replaced by trehalose group (D,T); however, FBS in the control freezing medium (freezing medium containing DMSO and FBS: D,S) was successfully substituted with KSR (replacement of FBS with KSR group). Both groups, trehalose supplementation in the freezing control group (D,S,T) and trehalose supplementation in the FBS replaced by KSR group (D,SR,T), did not show any difference compared with the freezing control, with respect to recovery rates. (B) Assessment of germ cell survival was demonstrated by proliferation capacity, which is determined through germ cell culture after freeze-thawing. All numerical values of proliferation capacity were presented as the percentage normalized to frozen control, which is the number of germ cells frozen in the control freezing medium containing DMSO, and FBS (control freezing medium, D,S). Removal of FBS from the freezing medium (D significantly reduced the proliferation capacity [49.1% ± 4.9%]). Replacement of FBS by KSR (D,SR) retained proliferation capacity (119.3% ± 5.4%), but the substitution with trehalose (D,T) significantly reduced proliferation capacity (68.6% ± 3.6%). Proliferation capacity was significantly increased when both groups were supplemented with trehalose (i.e., supplementation of trehalose in the control freezing medium [D,S,T] and the replacement of FBS by KSR medium [D,SR,T] [187.1% ± 11.9% and 160.8% ± 10.4%, respectively]) compared with control freezing group. There was no difference between the groups of D,S,T and D,SR,T. Values are means ± SEM (n = 6 independently established cultures for each treatment). Different letters indicate significant difference (P<.05) among groups.
Fertility and Sterility  , e5DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions