We thank Dr. Giulio Di Minin for derivation of phaESC lines and Dr. Remo Freimann for flow cytometry operation. We also acknowledge Ms. Mich&#232;le Schaffner and Mr. Thomas M. Hennek for technical support with embryo transfer. This work was supported by the Swiss National Science Foundation (grant 31003A_152814/1).

<ol>
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The authors have nothing to disclose.

Cloning of mammals by somatic cell nuclear transfer (SCNT) has been pioneered in the 1990s19,20,21. These developments followed cloning studies conducted 30 years earlier in amphibians22. The considerable delay reflects the difficulty of embryology and genomic imprinting in mammals. The development of mammalian SCNT is the basis for the application of haESC for substituting sperm, which is detailed in this protocol.
Cell cycle synchronization is an important factor for the success of SCNT23. This is also the case for injection of haESC in this protocol. Introducing a donor genome into a recipient requires the cell cycle phases to be matched to avoid chromosomal breakage or aneuploidies that would abrogate embryo development. Semi-cloning has the additional complexity that two genomes and a cytoplast need to be compatible. A previous report has demonstrated that injection of M-phase-arrested androgenetic haESCs yielded better developmental rates of semi-cloned embryos than injection of G1-phase haESCs into oocytes7. This report suggests M-phase as a suitable synchronization point for semi-cloning. Accordingly, phaESCs were mitotically arrested in metaphase with demecolcine and injected into the ooplasm of MII oocytes, which were naturally arrested in metaphase II of meiosis. Importantly, M-phase arrest can be achieved in mouse ESCs with high efficiency, providing excellent synchronization between donor and recipient cell cycles.
During mitosis, the nuclear membrane breaks down and a spindle forms to which the replicated chromosomes attach. After injection of M-phase DKO-phaESCs, sister chromatids are segregated into a pseudo polar body and the zygote7 (Figure 3E). Consequently, a single set of chromosomes from a DKO-phaESC contributes to the semi-cloned embryo. It is critical that the sister chromatids of DKO-phaESC chromosomes can be correctly segregated after injection. The plasma membrane of an intact DKO-phaESC prevents segregation into a pseudo polar body. We did indeed observe rare cases where the plasma membrane of DKO-phaESCs was not ruptured, and embryos exhibited intact DKO-phaESCs in the ooplasm after injection (Figure 3D). Therefore, care must be taken to remove the plasma membrane of DKO-phaESCs by pipetting. During injection, it is equally important to avoid the disruption of the meiotic spindle of the oocyte, which could lead to chromosome segregation defects and induce aneuploidy as well.
In mammals, genomic imprinting limits the application of phaESCs as sperm replacement. Parthenogenetic haESCs possess a maternal configuration of genomic imprints, whereas sperm possess a paternal configuration. Therefore, generation of full-term pups has not occurred after the injection of wildtype phaESCs as sperm replacement. To overcome this limitation, deletions of the IG- and H19-DMRs are engineered in phaESCs. Modification of imprinted expression at the maternal Igf2-H19 and Gtl2-Dlk1 loci is sufficient to change the configuration of genomic imprints for allowing the generation of semi-cloned mice with a frequency of over 5.1%, based on transferred 2-cell embryos. These observations suggest that targeting two imprinted genes switches the genome of phaESCs into a functional paternal configuration that can replace sperm in mice. Nevertheless, a permanent genetic modification of the phaESCs is required for this strategy. As an alternative strategy, androgenetic haESC can be considered. Androgenetic haESCs are derived from the sperm genome and possess paternal imprints. There have been reports that wildtype androgenetic haESCs contributed as sperm replacement to generate full-term pups at a frequency between 1.3% and 1.9% of transferred embryos4,7,24. Full-term pups have also been obtained by injecting androgenetic haESCs with deletions of the IG- and H19-DMRs at a frequency of 20.2% of transferred 2-cell embryos24. The increased efficiency of semi-cloning using modified androgenetic haESCs is likely because imprints can become unstable in culture. Imprinting defects are corrected by the genetic deletions of critical DMRs.
Considering the difficulty in introducing genetic modifications directly into the oocyte or sperm genomes, haESCs are a valuable tool for manipulating the parental genomes separately. Using haESCs as a substitute of sperm provides a remarkable advantage for genome editing in the mouse germline. A recent study has combined CRISPR/Cas9-based genome editing with the application of haESCs for the characterization of imprinting regions that are critical for embryonic development12. This study analyzed the role of the Rasgrf1-DMR in combination with the H19-and IG-DMRs in the development of bimaternal mice, and the function of 7 different DMRs in the development of bipaternal mice. The method for substituting haESCs for sperm formed the basis for genetic screening approaches for identifying key amino acids within the DND1 protein in primordial germ cell development and for identifying genes in bone development24,25,26. Studies on genomic imprinting and genetic screening to identify key factors in embryonic development are considerable&#160;approaches for the application of haESCs as gametic genomes.

In mammals, gametes are the only cells that transmit genetic information to the next generation. Fusion of an oocyte and a spermatozoon forms a diploid zygote that develops into an adult animal. Mutations in gametic genomes are thereby inherited by the offspring and drive genetic variation in species1. Introduction of mutations in the germline has been applied to produce genetically modified animals for diverse biological studies including the characterization of gene function and disease modelling. Both oocytes and spermatozoa are terminally differentiated and highly specialized cells that have ceased proliferation. Therefore, direct modification of gametes is technically difficult, and specialized approaches have been developed. Genetic modifications can be introduced into the mouse germline by the injection of genetically modified ESCs into blastocysts, where they integrate into the developing embryo and colonize the germline. Additionally, genetic modification of zygotes using genome editing approaches, including the CRISPR/Cas9 system, has become widely adopted2.
Recently, an outstanding approach has been reported, which applies haESCs as a substitute for a gametic genome3,4,5,6,7,8. HaESCs are stem cell lines derived from the inner cell mass of parthenogenetic or androgenetic haploid blastocysts and possess a single set of chromosomes4,7,9,10. It has been demonstrated that both parthenogenetic and androgenetic haESCs can contribute to the genome of semi-cloned mice after intracytoplasmic injection into oocytes. In contrast to other approaches, the genomes of haESCs can be directly modified in culture owing to their self-renewal capacity. Introducing genetic modifications into the germline by replacing sperm with haESCs is an important method for biological studies. It provides for a possibility to separately culture and manipulate the maternal or paternal genome, which are derived from parthenogenetic or androgenetic haESCs, respectively. HaESCs can then be used as gametic genome replacement, which is especially advantageous for studies of genomic imprinting, allele-specific expression, and parental specific processes.
In mice, both maternal and paternal genomic information is required for normal embryo development11. Therefore, full-term pups could not be obtained when wild-type parthenogenetic haESCs (phaESCs) were injected to replace the sperm genome5,8. To overcome the developmental block, genomic imprinting of the maternal genome of parthenogenetic haESCs needs to be corrected to a paternal configuration. This can be achieved by manipulation of the differentially methylated regions (DMRs). To date, targeted deletions of the H19-DMR, Gtl2-Dlk1 IG-DMR, and Rasgrf1-DMR have been studied to repress maternally expressed genes in phaESCs3,5,8,12. These studies demonstrated that deletions of both the H19-DMR and the IG-DMR are sufficient to convert a maternal into a paternal imprint configuration that can substitute for sperm chromosomes. Intracytoplasmic injection of phaESCs that carry the two DMR deletions into oocytes yielded semi-cloned pups with a frequency between 5.1% and 15.5% of transferred 2-cell embryos.
This protocol is based on the application of phaESCs with deletions of both H19-DMR and IG-DMR, which we term double-knockout phaESCs (DKO-phaESCs). We provide detailed instructions for the modification of genomic imprinting in phaESCs to establish DKO-phaESC lines, for the injection of DKO-phaESCs into oocytes as a substitute for a sperm genome, for culture of semi-cloned embryos to blastocysts, and for obtaining semi-cloned mice. This protocol is a reference for researchers who require precise and direct manipulation of the paternal genome and generation of semi-cloned embryos and mice.

<table>
	<tbody>
		<tr>
			<td>1.5 mLTube</td>
			<td>Eppendorf</td>
			<td>0030 125.150</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>15 ml Tube</td>
			<td>Greiner Bio-One</td>
			<td>188271</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>12-well plate</td>
			<td>Thermo Scientific</td>
			<td>150628</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>Thermo Scientific</td>
			<td>142475</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Thermo Scientific</td>
			<td>140675</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Thermo Scientific</td>
			<td>167008</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>Merck</td>
			<td>M6250</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>BSA fraction</td>
			<td>Gibco</td>
			<td>15260-037</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>Axon</td>
			<td>1386</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Demecolcine solution</td>
			<td>Merck</td>
			<td>D1925</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>41965-039</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DMEM / F-12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DR4 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>3208</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Merck</td>
			<td>E5134</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>biowest</td>
			<td>S1810-500</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Freezing medium</td>
			<td>amsbio</td>
			<td>11897</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>Merck</td>
			<td>G1890</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Hepes solution</td>
			<td>Gibco</td>
			<td>15630-056</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Irradiated MEF</td>
			<td>Gibco</td>
			<td>A34966</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>LIF (Leukemia inhibitory factor)</td>
			<td>(Homemade)</td>
			<td>-</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Lipofection reagent</td>
			<td>Invitrogen</td>
			<td>11668019</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>NDiff 227</td>
			<td>Takara</td>
			<td>Y40002</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>PD 0325901</td>
			<td>Axon</td>
			<td>1408</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>piggyBac plasmid carrying a CAG-EGFP transgene</td>
			<td>Addgene</td>
			<td>#40973</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>piggyBac transposase plasmid</td>
			<td>System Biosciences</td>
			<td>PB210PA-1</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>pX330 plasmid</td>
			<td>Addgene</td>
			<td>#42230</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>pX458 plasmid</td>
			<td>Addgene</td>
			<td>#48138</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco</td>
			<td>15090-046</td>
			<td>haESC culture</td>
		</tr>
		<tr>
			<td>DNA polymerase</td>
			<td>Thermo Scientific</td>
			<td>F549S</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>dNTP Mix</td>
			<td>Thermo Scientific</td>
			<td>R0192</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>100983</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck</td>
			<td>100317</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Merck</td>
			<td>109634</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Axon</td>
			<td>A3830</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>SDS</td>
			<td>Merck</td>
			<td>71729</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Merck</td>
			<td>106404</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>ThermoMixer</td>
			<td>Epeendorf</td>
			<td>5384000012</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Merck</td>
			<td>T1503</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>5 mL Tube</td>
			<td>Falcon</td>
			<td>352063</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>5 mL Tube with cell strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>MoFlo Astrios EQ</td>
			<td>Beckman Coulter</td>
			<td>B25982</td>
			<td>Flow cytometry</td>
		</tr>
		<tr>
			<td>1 mL syringe</td>
			<td>Codan</td>
			<td>62.1640</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>30-gauge 1/2 inch needle</td>
			<td>Merck</td>
			<td>Z192362-100EA</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>B6D2F1 mouse</td>
			<td>The Jackson Laboratory</td>
			<td>100006</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>MSD animal health</td>
			<td>49451</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Gibco</td>
			<td>10010015</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>Avivasysbio</td>
			<td>OPPA1037 5000 IU</td>
			<td>Superovulation</td>
		</tr>
		<tr>
			<td>10-cm dish</td>
			<td>Thermo Scientific</td>
			<td>150350</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>4-well plate</td>
			<td>Thermo Scientific</td>
			<td>179830</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>6 cm dish</td>
			<td>Thermo Scientific</td>
			<td>150288</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Center-well dish</td>
			<td>Corning</td>
			<td>3260</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Digital rocker</td>
			<td>Thermo Scientific</td>
			<td>88880020</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>AppliChem</td>
			<td>A0878</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Merck</td>
			<td>H4272</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>Merck</td>
			<td>MR-020P-5F</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>M16 medium</td>
			<td>Merck</td>
			<td>M7292</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>Merck</td>
			<td>M7167</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Merck</td>
			<td>M8410</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Glass capillary</td>
			<td>Merck</td>
			<td>P1049-1PAK</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SMZ745</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>Strontium chloride</td>
			<td>Merck</td>
			<td>13909</td>
			<td>Embryo manipulation</td>
		</tr>
		<tr>
			<td>5ml syringe</td>
			<td>Codan</td>
			<td>62.5607</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Borosilicate glass cappilary</td>
			<td>Science product</td>
			<td>GB100T-8P</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>CellTram 4r Oil</td>
			<td>Eppendorf</td>
			<td>5196000030</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Fluorinert FC-770</td>
			<td>Merck</td>
			<td>F3556</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Holding pipette</td>
			<td>BioMedical Instruments</td>
			<td>-</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Nikon</td>
			<td>Eclipse Ti-U</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microforge</td>
			<td>Narishige</td>
			<td>MF-900</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microinjection pipette</td>
			<td>BioMedical Instruments</td>
			<td>-</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Microlaoder tips</td>
			<td>Eppendorf</td>
			<td>5252 956.003</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Micromanipulaor arms</td>
			<td>Narishige</td>
			<td>NT-88-V3</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P-1000</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>PiezoXpert</td>
			<td>Eppendorf</td>
			<td>5194000016</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>PVP (Polyvinylpyrrolidine)</td>
			<td>Merck</td>
			<td>PVP360</td>
			<td>Microinjection</td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>SARSTEDT</td>
			<td>83.1826.001</td>
			<td>Microinjection</td>
		</tr>
	</tbody>
</table>

application of mouse parthenogenetic haploid embryonic stem cells as a substitute of sperm

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an oocyte by a spermatozoon creates a totipotent zygote. Recently, several publications have reported that haploid embryonic stem cells (haESCs) can be a substitute for gametic genomes and contribute to embryos, which develop into mice. Here, we present a protocol to apply parthenogenetic haESCs as a substitute of sperm to construct embryos by intracytoplasmic injection into oocytes. This protocol consists of steps for preparing haESCs as sperm replacement, for injection of haESC chromosomes into oocytes, and for culture of semi-cloned embryos. The embryos can yield fertile semi-cloned mice after embryo transfer. Using haESCs as sperm replacement facilitates genome editing in the germline, studies of embryonic development, and investigation of genomic imprinting.

The purpose of this protocol is to apply haESCs as a substitute of sperm to obtain semi-cloned embryos and mice. For this purpose, a DKO-phaESC line carrying the CAG-EGFP transgene was generated and used for intracytoplasmic injection into MII oocytes. To obtain a suitable phaESC line with a paternal imprint configuration, we performed genetic engineering using Cas9 nucleases. A haploid ESC line contains haploid and diploid cells that arise due to an inherent tendency of haploid ESCs for diploidization10. A haploid chromosome set is a prerequisite for successful replacement of the sperm genome. DNA content analysis by flow cytometry shows the distribution of haploid and diploid cells at G0/G1-, S-, and G2/M-phases (Figure 2A).
To establish the DKO-phaESC lines, wildtype phaESC lines were transfected with piggyBac transposon constructs for stable transgenic EGFP expression and with CRISPR/Cas9 plasmids for obtaining deletions of the H19- and IG-DMRs. To exclude diploid ESCs and isolate only haploid ESCs expressing EGFP, a specific sort gate was defined (Figure 2A). Single haploid EGFP-positive cells were then plated into individual wells of 96-well plates to obtain sub-clones. MEF feeders were used to increase the plating efficiency and survival of the transfected phaESCs. After expansion of the cultures, an initial round of genotyping was carried out by PCR to identify sub-clones that carry deletions of both DMRs. After MEF feeders had been removed from the cultures, a second genotyping was performed to confirm the absence of wildtype alleles at the H19- and IG-DMRs (Figure 2B). From a total of 135 sub-clones, we obtained 5 haploid DKO-phESC lines that carried the deleted alleles and were free of wildtype alleles of both the H19-DMR and the IG-DMR.
A CAG-EGFP transgene was introduced into DKO-phaESCs to study their contribution to semi-cloned embryos by visualization of green fluorescence under a microscope (Figure 3A). For the intracytoplasmic injection, DKO-phaESCs were treated with demecolcine to arrest them in M-phase. Thus, the cell cycle of DKO-phaESCs was synchronized with that of MII oocytes. Flow cytometry analysis showed 2 populations corresponding to G2/M phase arrested haploid (2n) and diploid cells (4n) after the treatment with demecolcine (Figure 3B). Absence of the 1n haploid peak indicated that the cell cycle arrest was largely complete. M-phase haploid ESCs were then sorted and injected into oocytes. For this, a single DKO-phaESC was loaded into the microinjection pipette and injected into the cytoplasm of a MII oocyte (Figure 3C). The plasma membrane of the DKO-phaESC was ruptured by pipetting into the tip of the microinjection pipette.
After the injection, EGFP expression was rarely detected in the constructed semi-cloned embryos as the cytoplasm of DKO-phaESCs had dispersed in the large cytoplasm of the oocyte (Figure 3D). In rare cases, a round spot of intense EGFP expression could be observed within the ooplasm. This observation was likely caused by the inadvertent injection of intact DKO-phaESCs. Failure to rupture the DKO-phaESC cell membrane is likely not compatible with further embryo development and should be avoided. One hour after the injection, embryos were activated by treatment with strontium chloride18. Six hours after the initiation of activation, up to 3 polar bodies were observed under the microscope (Figure 3E). These polar bodies correspond to the first and second polar bodies of the oocyte and a pseudo polar body from the DKO-phaESC7. In addition, two pronuclei were observed under a differential interference contrast microscope, which resembled the pronuclear stage of zygotes after normal fertilization with sperm.
To demonstrate developmental competence, semi-cloned embryos were cultured to the blastocyst stage (Figure 5A). Furthermore, a full-term mouse was obtained after transferring semi-cloned 2-cell stage embryos into the oviducts of a recipient female (Figure 5B). As expected, the mouse derived from a semi-cloned embryo was a female as neither oocytes nor oocyte-derived phaESCs carry a Y chromosome. The semi-cloned mouse was overtly normal and produced healthy offspring when mated with a wildtype Swiss Webster male. Until now, the semi-cloned mouse has been alive for over 600 days without any apparent health problems.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61999/61999fig01v3.jpg" /> 
Figure 1: Overview of the application of DKO-phaESCs as sperm replacement.&#160;(A) A time frame of the procedures of the protocol. (B) The scheme shows the steps to establish DKO-phaESC lines (steps 1&#8211;6). (C) The scheme shows the steps to construct semi-cloned embryos by intracytoplasmic injection of a DKO-phaESC into a MII oocyte (steps 7&#8211;14). Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell; FACS = fluorescence-activated cell sorting; MEF = mouse embryonic fibroblast. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61999/61999fig02v2.jpg" /> 
Figure 2: Establishment of DKO-phaESC lines by CRISPR/Cas9-mediated deletions of H19- and IG-DMRs.&#160;(A) Flow cytometry analysis of phaESCs after transfection with piggyBac plasmids for stable EGFP expression and with 4 CRISPR/Cas9 plasmids. Non-transfected phaESCs are shown as control. The DNA profile (top) shows the cell cycle distribution of haploid and diploid cells. G1/S-phase haploid cells expressing EGFP are indicated by the green gate (bottom, right). (B) Genotyping of phaESC sub-clones that were grown on MEFs (first genotyping) and after removal of MEFs (second genotyping). Sub-cloned phaESC lines 1, 2, 3, and 4 represent wildtype cells, cells with a H19-DMR deletion, with an IG-DMR deletion, and with combined H19- and IG-DMR deletions, respectively. DKO-phaESC lines 5&#8211;8 possess both H19-DMR and IG-DMR deletions and are free from wildtype alleles. Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell; DMR = differentially methylated region; MEF = mouse embryonic fibroblast; EGFP = enhanced green fluorescent protein; WT = wildtype. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61999/61999fig03v3.jpg" /> 
Figure 3: Injection of mitotically arrested DKO-phaESCs into MII oocytes.&#160;(A) Morphology of a DKO-phaESC culture that carries a CAG-EGFP transgene and deletions of the H19- and IG-DMRs; scale bar = 50 &#181;m. (B) Representative flow cytometry analysis of DKO-phaESCs after arrest with demecolcine for 8 h. DKO-phaESCs without demecolcine treatment are shown as control. (C) Morphology of DKO-phaESCs in the microinjection pipette. A single intact DKO-phaESC before (left) and after (right) rupturing the plasma membrane by pipetting is shown; scale bar = 20 &#181;m. (D) Constructed embryos at 1 h after injection of DKO-phaESCs into MII oocytes. A merged image of EGFP fluorescence and bright field is shown. Black arrowheads indicate round spots of intense EGFP expression after injection of intact DKO-phaESCs, which should be avoided; scale bar = 50 &#181;m. (E) A differential interference contrast image of semi-cloned embryos 6 h after the initiation of activation with strontium chloride is shown. White arrowheads indicate embryos with 3 polar bodies including one pseudo polar body from the injected DKO-phaESC; scale bar = 50 &#181;m. Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell; EGFP = enhanced green fluorescent protein. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61999/61999fig04.jpg" /> 
Figure 4: Scheme of the setup for intracytoplasmic injection of DKO-phaESCs into oocytes.&#160;(A) The arrangement of an injection pipette, a holding pipette, and an oocyte in the injection chamber is shown. &#945;, bend&#160;angle of the microinjection pipette. (B) Layout of the drops in the micromanipulation dish for intracytoplasmic injection. Abbreviations: DKO = double-knockout; phaESC = parthogenetic haploid embryonic stem cell. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61999/61999fig05.jpg" /> 
Figure 5: Development of semi-cloned embryos.&#160;(A) Preimplantation development of semi-cloned embryos in vitro. EGFP expression is initially observed in four-cell embryos at day 2 after intracytoplasmic injection. At day 4, intense EGFP expression can be observed in blastocysts; scale bar = 100 &#181;m. (B) A semi-cloned mouse obtained after 2-cell embryo transfer to a recipient female. At 74 dpp, the semi-cloned mouse (arrowhead) delivered her first pups (asterisk) by natural birth after mating with a wildtype Swiss Webster male. Semi-cloned embryos and the semi-cloned mouse shown in this figure are identical with ones reported in Aizawa et al.3. Abbreviations: EGFP = enhanced green fluorescent protein; .dpp, days post-partum. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/61999/61999fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Gelatin solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>500 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>-</td>
			<td>1 g</td>
			<td>0.2%</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Haploid embryonic stem cell (HaESC) medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>NDiff 227</td>
			<td>-</td>
			<td>10 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>CHIR 99021</td>
			<td>10 mM</td>
			<td>3 &#181;L</td>
			<td>3 &#181;M</td>
		</tr>
		<tr>
			<td>PD 0325901</td>
			<td>10 mM</td>
			<td>1 &#181;L</td>
			<td>1 &#181;M</td>
		</tr>
		<tr>
			<td>LIF</td>
			<td>1 x 106 IU/mL</td>
			<td>10 &#181;L</td>
			<td>1,000 IU/mL</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100x</td>
			<td>100 &#181;L</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">HaESC maintenance buffer</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>HaESC medium</td>
			<td>-</td>
			<td>1 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>HEPES solution</td>
			<td>1 M</td>
			<td>20 &#181;L</td>
			<td>20 mM</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Wash buffer</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>DMEM / F-12</td>
			<td>-</td>
			<td>100 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BSA fraction</td>
			<td>7.5%</td>
			<td>7.1 mL</td>
			<td>0.5%</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">MEF medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>-</td>
			<td>500 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>-</td>
			<td>56 mL</td>
			<td>10%</td>
		</tr>
		<tr>
			<td>&#946;-Mercaptoethanol</td>
			<td>14.3 mol/L</td>
			<td>3.9 &#181;L</td>
			<td>100 &#181;M</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>100x</td>
			<td>5.6 mL</td>
			<td>1x</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Lysis buffer</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>-</td>
			<td>8.25 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Tris-HCl (pH 8.5)</td>
			<td>1 M</td>
			<td>1 mL</td>
			<td>100 mM</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>0.5 M</td>
			<td>100 &#181;L</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>SDS solution</td>
			<td>10%</td>
			<td>200 &#181;L</td>
			<td>0.2%</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>5 M</td>
			<td>400 &#181;L</td>
			<td>200 mM</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>20 mg/mL</td>
			<td>50 &#181;L</td>
			<td>100 &#181;g/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Activation medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>KSOM</td>
			<td>-</td>
			<td>1 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Strontium chloride</td>
			<td>1 M</td>
			<td>5 &#181;L</td>
			<td>5 mM</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>0.5 M, pH 8.0</td>
			<td>4 &#181;L</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">PVP solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>-</td>
			<td>5 mL</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PVP</td>
			<td>-</td>
			<td>0.6 g</td>
			<td>12%</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">PMSG solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>-</td>
			<td>450 &#181;L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>PMSG</td>
			<td>500 IU/mL</td>
			<td>50 &#181;L</td>
			<td>50 IU/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">hCG solution</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>-</td>
			<td>450 &#181;L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>hCG</td>
			<td>500 IU/mL</td>
			<td>50 &#181;L</td>
			<td>50 IU/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Hyaluronidase medium</td>
		</tr>
		<tr>
			<td>Component</td>
			<td>Stock concentration</td>
			<td>Volume / weight</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>M2 medium</td>
			<td>-</td>
			<td>380 &#181;L</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>10 mg/mL</td>
			<td>20 &#181;L</td>
			<td>0.5 mg/mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Abbreviations: LIF = leukemia inhibitory factor; MEF = mouse embryonic fibroblast;</td>
		</tr>
		<tr>
			<td colspan="4">FBS = fetal bovine serum; DMEM = Dulbecco&#39;s Modified Eagle Medium; BSA = bovine serum albumin;</td>
		</tr>
		<tr>
			<td colspan="4">EDTA = ethylenediaminetetraacetic acid; SDS = sodium dodecylsulfate; EGTA = ethylene-bis(oxyethylenenitrilo)tetraacetic acid;</td>
		</tr>
		<tr>
			<td colspan="4">PBS = phosphate-buffered saline; PVP = polyvinylpyrrolidone; hCG = human chorionic gonadotropin;</td>
		</tr>
		<tr>
			<td colspan="4">PMSG = pregnant mare serum gonadotropin.</td>
		</tr>
	</tbody>
</table>
Table 1: Recipe of medium, buffer, and solution.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name</td>
			<td colspan="2">Sequence (5&#39; to 3&#39;)</td>
			<td>Application</td>
		</tr>
		<tr>
			<td>H19-DMR-P1</td>
			<td colspan="2">GTG GTT AGT TCT ATA TGG GG</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>H19-DMR-P2</td>
			<td colspan="2">AGA TGG GGT CAT TCT TTT CC</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>H19-DMR-P3</td>
			<td colspan="2">TCT TAC AGT CTG GTC TTG GT</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>IG-DMR-P1</td>
			<td colspan="2">TGT GCA GCA GCA AAG CTA AG</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>IG-DMR-P2</td>
			<td colspan="2">CCA CAA AAA CCT CCC TTT CA</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>IG-DMR-P3</td>
			<td colspan="2">ATA CGA TAC GGC AAC CAA CG</td>
			<td>Genotyping</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA1-F</td>
			<td colspan="2">CAC CCA TGA ACT CAG AAG AGA CTG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA1-R</td>
			<td colspan="2">AAA CCA GTC TCT TCT GAG TTC ATG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA2-F</td>
			<td colspan="2">CAC CAG GTG AGA ACC ACT GCT GAG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>H19-DMR-gRNA2-R</td>
			<td colspan="2">AAA CCT CAG CAG TGG TTC TCA CCT</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA1-F</td>
			<td colspan="2">CAC CCG TAC AGA GCT CCA TGG CAC</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA1-R</td>
			<td colspan="2">AAA CGT GCC ATG GAG CTC TGT ACG</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA2-F</td>
			<td colspan="2">CAC CCT GCT TAG AGG TAC TAC GCT</td>
			<td>gRNA</td>
		</tr>
		<tr>
			<td>IG-DMR-gRNA2-R</td>
			<td colspan="2">AAA CAG CGT AGT ACC TCT AAG CAG</td>
			<td>gRNA</td>
		</tr>
	</tbody>
</table>
Table 2: List of oligonucleotides.

Watch this Scientific Journal Video about Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm at JoVE.com

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

In organisms with sexual reproduction, germ cells are the source of totipotent cells that develop into new individuals. In mice, fertilization of an ...

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4.5K Views.  ETH Zurich. This article aims to demonstrate the use of parthenogenetic haploid embryonic stem cells as a substitute for sperm for the construction of semi-cloned embryos.

Video: Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

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Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, and is necessary for the emergence of definitive HSPC from the murine extra embryonic yolk sac, placenta, umbilical vessels, and the embryonic aorta-gonad-mesonephros (AGM) region. The transient nature and small size of this cell population renders its reproducible isolation for careful quantification and experimental applications technically difficult. We have established a fluorescence-activated cell sorting (FACS)-based protocol for simultaneous isolation of hemogenic endothelial cells and HSPC during their peak generation times in the yolk sac and AGM. We demonstrate methods for dissection of yolk sac and AGM tissues from mouse embryos, and we present optimized tissue digestion and antibody conjugation conditions for maximal cell survival prior to identification and retrieval via FACS. Representative FACS analysis plots are shown that identify the hemogenic endothelial cell and HSPC phenotypes, and describe a methylcellulose-based assay for evaluating their blood forming potential on a clonal level.

Hematopoietic stem and progenitor cells (HSPC) derive from specialized (hemogenic) endothelial cells during development, yet little is known about the process by which some endothelial cells specify to become blood forming. We demonstrate a flow-cytometry based method allowing simultaneous isolation of hemogenic endothelial cells and HSPC from murine embryonic tissues.

The specification of hemogenic endothelial cells from embryonic vascular endothelium occurs during brief developmental periods within distinct tissues, ...

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

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Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

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Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

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Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous transplants to treat hematological and malignant disorders. Hemogenic endothelium (HE) is an amenable platform to produce functional HSPCs by transcription factors or to induce lymphocytes in a robust manner. However, current methods to derive HE are either costly or variable in yield. In this protocol, we established a cost-effective and precise method to derive HE within 4 days in a feeder- and xeno-free defined condition. The resultant HE underwent an endothelial-to-hematopoietic transition and produced CD34+CD45+ clonogenic hematopoietic progenitors. The potential capacity of our platform for generating functional HSPCs will have broad applications in disease modeling and screening for therapeutic compounds.

Here, we present a protocol to precisely form hemogenic endothelium from human pluripotent stem cells in a feeder- and xeno-free condition. This method will have broad applications in disease modeling and screening for therapeutic compounds.

Deriving functional hematopoietic stem and progenitor cells (HSPCs) from human pluripotent stem cells (PSCs) have been an elusive goal for autologous ...

Application of Mouse Parthenogenetic Haploid Embryonic Stem Cells as a Substitute of Sperm

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Chapters in this video

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Isolation of Murine Embryonic Hemogenic Endothelial Cells

The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

Hemogenic Endothelium Differentiation from Human Pluripotent Stem Cells in A Feeder- and Xeno-free Defined Condition