BLASTOCYST DEVELOPMENT

AFTER
FERTILIZATION WITH IN
VITRO SPERMATIDS
DERIVED FROM NONHUMAN
PRIMATE
EMBRYONIC STEM CELLS

Viktorie Laštůvková
BACKGROUND
Global health issue -“Sperm crisis”
Posibility of producing spermatogonia-like cells,
primary and secondary spermatocyte-like cells,
haploid spermatid-like cells
Mouse - live offsprrings from gamete-like cells
Diferencies between rodent and human ➔ nonhuman
primate (NHP)
POINTS OF THE RESEARCH
Green fluorescent protein-labeled, macaque rhesus nonhuman
primate embryonic stem cells differentiation into advanced male
germ cell lineages
In vitro-derived round spermatid-like cells
Intracytoplasmic sperm(atid) injection (ICSI)
Fertilization
Preimplantation embryo development (from zygote to blastocyst)
METHODS
Cultivation in SSC medium (10 Days)
RNA isolation and sequencing (Differential gene expression
analysis)
MII oocyte colection (hormonal stimulation), spermatide isolation
ICSI
FISH analyse for diploidy\haploidy determination
Immunostaining for detection postmeiotic spermatid markers
Fluorescence activated cell sorting
And more…
METHODS

Easley 4th CA, Phillips BT, McGuire MM, Barringer JM, Valli H, Hermann BP, et al. Direct differentiation of human pluripotent stem cells into
haploid sper- matogenic cells. Cell Rep 2012;2:440–6.
RESULTS - SPERMATIDE DIFFERENTIATION
RESULTS - FERTILIZATION
RESULTS - PREIMPLANTATION EMBRYO DEVELOPMENT

A B C

D E F
RESULTS
1,875 upregulated and 1,431 downregulated genes in new
differentiations
Upregulation of genes involved in spermatogenesis, including
PAX6, GFRA1, DMRT1, and ZBTB16
Downregulation of genes critical for maintaining pluripo- tency,
POU5F1, SPP1, and MYC
Increased VASA protein expression
Haploid cell population averaging approximately 5.3% across
multiple differentiations
DISCUSION
Functional haploid spermatids can be differentiated
from NHP pluripotent stem cells completely in vitro
Haploid spermatids derived from NHP pluripotent
stem cells can fertilize MII oocyte
Round spermatids are immature
Optimized conditions
Resulting blastocysts are not of parthenogenetic
origin
H2B-GFP expression in the nuclei of cells
throughout the developing embryo
REFERENCES
Blastocyst development after fertilization with in vitro spermatids
derived from nonhuman primate embryonic stem cells. Khampang,
Sujittra et al. F&S Science, Volume 2, Issue 4, 365 - 375
Easley 4th CA, Phillips BT, McGuire MM, Barringer JM, Valli H,
Hermann BP, et al. Direct differentiation of human pluripotent stem
cells into haploid sper- matogenic cells. Cell Rep 2012;2:440–6.
 THANK YOU
FOR YOUR
ATTENTION!

Viktorie Laštůvková
MATERIALS
for pluripotent stem cell lineage

SSC medium

STO-feeder cells -line established from mouse SIM embryonic fibroblasts, have been frequently used for
establishing embryonic stem cells and maintaining them in an undifferentiated state.

Blood gas mixture - (5% CO2, 5% O2, 90% N2 gas)

Differentiation - glial-derived neurotrophic factor, fibroblast growth

factor
Spermatides - TET3 protein, SCF (sperm cytoplasmatic Factor)
Ferthilized embryos - HECM-9 medium (Hamster embryo culture
medium, 10 hours)
WHAT SSC MEDIUM CONTAINS?
SSC = mouse spermatogonial stem cell medium, every 10 hours
MEM alpha (Thermo Fisher), 0.2% bovine serum albumin
(Sigma), 5 mg/mL of insulin (Sigma), 10 mg\mL of
transferrin (Sigma), 60 mM of putrescine (Sigma), 2 mM of
L-glutamine (Invitrogen), 50 mM of beta-merca- ptoethanol
(Sigma), 1 ng/mL of human basic fibroblast growth factor
(R&D Systems), 20 ng/mL of glial cell line-derived
neurotrophic factor (R&D Systems), 30 nM of sodium
selenite (Sigma), 2.36 mM of palmitic acid (Sigma), 0.21 mM
of palmitoleic acid (Sigma), 0.88 mM of stearic acid (Sigma),
1.02 mM of oleic acid (Sigma), 2.71 mM of linoleic acid
(Sigma), 0.43 mM of linolenic acid (Sigma),10 mM of 4-(2-
hydroxyethyl)-1-piperazine - ethane- sulfonic acid (HEPES
[Sigma]), 0.5 penicillin /streptomycin (Thermo Fisher)