Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice

Posted on
generation-of-exogenous-kidneys-via-crispr/cas9-mediated-blastocyst-complementation-targeting-osr1-gene-in-mice
Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice

Introduction

In vitro culture and production of desired organs are currently one of the potential solutions to the global deficit of transplantable organs. Due to the limited diffusion range of essential molecules, malformations of blood vessels and neurons are frequently found, and hence, malfunctions of the harvested organs with abnormal tissue cells are observed, compared to those of normal organs1. Therefore, searching for new ways to provide enough and functionally normal organs is essential.

Among all the different varieties of methods, one alternative to generate potential organs is the injection of wild-type ES cells into specific organ-disabled blastocysts, followed by embryo transfer. For instance, Kobayashi et al. (2010) successfully produced rat pancreas by injecting wild-type rat ES cells into Pdx1-/- mouse blastocysts, the pancreatogenesis-disabled embryos2. The injected ES cells compensated for the missing part of the mouse pancreas, indicating the possibility of xeno-organogenesis by using such blastocyst complementation technique. Based on this concept, Matsunari et al. (2013) injected the cloned pig blastomeres carrying the humanized Kusabira-Orange (huKO) fluorescent protein into pancreatogenesis-disabled pig embryos, and also successfully generated an entire pancreas consisting of huKO cells inside the Pdx1-/- pig3. These outcomes hold great promise for the production of organs from specific organ-disabled animals, but there are still obstacles ahead. Currently, we are in need of a comprehensive understanding of intercellular interactions during chimera formation and the complemented model for the desired organ, for instance, the kidney. Usui et al. (2012) have tried to generate allo-kidneys by injecting mouse pluripotent stem (PS) cells into Sall1-/- mouse blastocysts4. Although mouse kidneys with mostly wild-type donor cells have been produced, however, all pups failed to survive to adulthood for unknown reasons. Apparently, the genes associated with nephrogenesis are far more complicated than those for pancreatogenesis. In addition to the complication of kidney development, interactions between two sources of cells during development could even further aggravate the low successful rate of blastocyst complementation. At the genetic level, Pdx1-/- and Sall1-/- have different impacts on knockout mouse development; Sall1-/- not only disabled nephrogenesis but also affected the olfactory bulb development and suckling ability of fetuses5,6. Therefore, it is challenging to develop better strategies for kidney- and other organ-disabled models without compromising other tissue functions.

In mammals, kidney formation is closely associated with the development of the reproductive system. After the development of pronephros and mesonephros, the ureteric bud at the metanephros interacts with the intermediate mesoderm that induces the formation of the metanephrogenic blastema; the ureteric bud develops into ductogenic lineage and metanephrogenic blastema that subsequently undergoes mesenchyme-epithelium transition (MET) to become the nephrogenic lineage of the kidney7. Therefore, it is possible to generate a kidney-disabled embryo model by disrupting the induction pathway of metanephrogenic blastema. In previous studies, several critical genes, such as Gdnf8, Eya19, and Odd1 (a.k.a. Osr1)10,11, have been identified during nephrogenesis. Osr1 is the upstream gene that regulates key signaling pathways associated with the expressions of Gdnf, Eya1, and Sall1 for early nephric duct elongation and kidney induction. We hypothesize that knocking out Osr1 should completely disrupt the development of the whole urinary system beyond the formation of mesonephric ducts. To generate totally complemented kidneys, the host cells from the knockout model need to be completely “clean” downstream of the developing ureteric bud. Therefore, in this study, we test the feasibility of using CRISPR/Cas9-mediated gene knockout procedure to target Osr1, accompanied by blastocyst complementation technique for the production of mouse exogenous kidneys.

Previously, most of the blastocyst complementation studies used either somatic cell nuclear transfer (SCNT) and/or heterogeneous breeding to create organ-disabled blastocysts. Although SCNT is a reliable method to produce genetically identical embryos and offspring, these clones can have multiple defects, including poor quality, delayed development, and impaired differentiation potential, mainly caused by the incomplete reprogramming12,13,14. Moreover, to become skillful, researchers, however, require an intensive and lengthy training procedure to minimize injury to the manipulated embryos. Previous studies have applied the CRISPR/Cas9 system to produce complementary pancreas, heart, eyes, and forebrain with various degrees of chimerism in the target organ15,16,17. In consideration of the high cost and epigenetic defects of SCNT, we hypothesized that CRISPR/Cas9-mediated gene knockout by pronuclear injection in combination with blastocyst complementation might be a better solution to circumvent SCNT pitfalls as well as to save tremendous time and fund investments. Therefore, to produce exogenous kidneys, we decided to first knock out Osr1 by microinjection of CRISPR/Cas9 constructs, followed by the classical blastocyst complementation procedure by injection of wild-type EGFP+ ES cells as depicted in Fig. 1.

Fig. 1
figure 1

The schematic illustration for the present study is shown along with the developmental stages of embryos. The sgRNA constructs are injected into the pronuclear stage mouse embryos, followed by various tests for the complementation efficiencies. (1) Blastocyst embryos and ES cells are collected for genotyping the knockout efficiency. (2) The injected embryos are transferred into pseudo-pregnant surrogates, and E12.5–13.5.5 embryos are harvested for genotyping to confirm the suitability of kidney complementation. (3) The sgRNA-injected embryos were further transplanted with EGFP+ ES cells to produce exogenous kidneys

Full size image

Results

CRISPR/Cas9-mediated knockout embryos by using multiple guide RNAs

CRISPR/Cas9-mediated blastocyst complementation has been proven to be an efficient way to produce cellular-complemented animals, but the efficiency may vary between genes and construct designs. We first test the efficiency of our sgRNAs on Osr1. In our initial tests, we used two and three sgRNAs to target the exon 2 of Osr1. Exon 2 is the first coding exon of Osr1, and it has been proven that knocking out exon 2 of Osr1 results in dysfunction of the encoded protein10. The Cas9 RNPs were premixed and injected into the mouse pronucleus. The injected zygotes were cultured to the blastocyst stage and collected for the knockout efficiency test. For genotyping the blastocysts derived from two sgRNA-injected pronuclear stage embryos, a total of 104 blastocyst embryos were used, of which 69 (66.4%) were found with at least one 287-bp band (wildtype or heterozygous), and 35 (33.6%) were found no 287-bp band (deletion on both alleles) (Fig. 2). We acknowledge that capillary electrophoresis-based analysis may not definitively distinguish between a true heterozygous state and mosaicism. However, for the purpose of our study, any embryo showing at least one functional allele was categorized as non-knockout. Based on the preliminary tests, we decided to further enhance knockout efficiencies by using three sgRNA-injected ES cells for blastocyst complementation.

In the 10 ES cell lines we established, four of them showed biallelic knockout on Osr1; two of them had a deletion on one allele, and four of them remained wild type (Fig. 3A). Furthermore, sequencing the six cell lines with deletions confirmed that the indel sequences predominantly corresponded to the three sgRNA target sites, thereby validating their specific and effective targeting of the Osr1 gene (Fig. 3B). With the effect of the three-sgRNA system confirmed in ES cells, we proceeded with our blastocyst complementation experiments using this optimized protocol.

Fig. 2
figure 2

Efficacies of CRISPR/Cas9 gene editing targeting Osr1 with two sgRNA constructs. (A) Scheme of the sgRNA and primers targeted positions at Osr1. (B) An example of the capillary electrophoresis for blastocysts after injection of Cas9 protein along with two sgRNAs, Osr1a and Osr1b. The Osr1-WT-1 is the wild-type blastocyst. PCR amplification using Primer 2 and Primer 3 yielded a single 287-bp band from the wild-type blastocyst. Those blastocysts were evaluated by whether they have bands equal to the wild-type embryo (287-bp) or not. If it has only one 287-bp band, we count it as a wild-type. If it has one band at 287 bp and the other drops below it, then it is heterozygous. If it only has bands below 287 bp, it indicates having deletions on both alleles. In this batch of blastocysts, numbers 3, 5, 9, 11, 12, 13, 14, 15, 16, and 18 are found no deletion; number 19 is heterozygous; numbers 1, 2, 4, 6, 7, 8, 10, and 17 are biallelic knockouts. (C) The efficiencies of gene editing with two sgRNAs (Osr1a and Osr1b)

Full size image

Fig. 3
figure 3

Establishment of Osr1 knockout mouse ES cells derived from the blastocyst embryos developed from Cas9 protein and 3 sgRNAs injected mouse zygotes. (A) Electrophoresis result of 10 possible Osr1 knockout ES cell lines. Primer 2 and Primer 5 were used to amplify 929-bp products from wild-type ES cells. If it has only one 929-bp band, we count it as wild-type. If it has one band at 929 bp and the other drops below it, then it is heterozygous. If it only has bands below 929 bp, it indicates deletions on both alleles. Therefore, ES cell (Lane) #1, 5, 7, and 9 are wild-type; #2, 6 are heterozygous; #3, 4, 8, and 10 are biallelic knockout. The full gel image can be found in the supplementary information (Fig. S1). (B) Alignment of the sequencing results with the Osr1 gene. The numbers 1 to 10 correspond to Lane 1 to Lane 10 in (A)

Full size image

Lethality of Osr1 knockout on E13.5 mouse embryos

The effects of the three sgRNAs on the targeted Osr1 were tested. After the transfer of the blastocysts derived from the three sgRNA-injected pronuclear stage embryos, we found that spontaneous resorption of the implanted embryos occurred from E12.5 to E13.5, with 72.3% being degenerated embryos (Fig. 4; Table 1). We further examined the harvested embryos between E12.5 and E13.5 and found that most of the embryos were degenerated, with some intact embryos that lacked a metanephros tube and kidney formation being harvested (Fig. 5). This suggested that our CRISPR/Cas9 constructs could knock out the whole kidneys in the mouse system.

Table 1 In vivo development of mouse embryos after pronuclear microinjection with CRISPR/Cas9 constructs targeting Osr1 gene followed by embryo transfer

Full size table

Fig. 4
figure 4

In vivo development of the mouse fetus (E13.5). (A) The pronuclear stage embryos were microinjected with CRISPR/Cas9 constructs targeting the Osr1 gene. After embryo transfer, the implanted uterine horns are harvested on day 13.5, showing mostly degenerated fetuses. Scale bar: 2 mm. (B) Most of the fetuses show various degrees of degeneration. Scale bar: 1 mm

Full size image

Fig. 5
figure 5

Histological examination (HE staining) of E12.5 mouse fetuses pronuclear injected with CRISPR/Cas9 constructs targeting Osr1 gene. (A) A wild-type control fetus (without construct injection). (B) A CRISPR/Cas9 construct-injected fetus shows no observable kidney development (red dotted circle). h: heart; li: liver; st: stomach; ki: kidney. Scale bar: 200 μm

Full size image

Rare Osr1-complemented kidneys supported fetal development

After confirming the efficiency of our CRISPR/Cas9 constructs, we proceeded to generate exogenous kidneys through CRISPR/Cas9-mediated blastocyst complementation by the injection of EGFP+ ES cells, with wild-type Osr1. It resulted in the low birth rate (4 pups out of 264 embryos transferred, 1.5%) after embryo transfer, although implantation sites with the fetal vesicles in the recipient uterus (E13 and E15) were observed. We then tried to harvest the embryos between E12.5 to E15.5, from which most of the embryos were found to be degenerated during E13.5 to E15.5, and no degenerated embryos at E12.5 were observed (Table 2). The EGFP signals were observable in the degenerated fetuses or tissues, indicating the formation of chimeras in the complemented fetuses (Fig. 6). Retarded or degenerated embryos can be found in both the chimeric and non-chimeric fetuses (Figs. 6C and 7). Apparently, fetuses degenerated and died, partly because of the incapability of the injected wild-type ES cells to compensate for the deficient organs.

We closely examined the E12.5 chimeras; at this stage, the nephric ducts were already formed, and most of their mesonephric and metanephric ducts scarcely showed EGFP+ cells (Fig. 8A and B), except for one fetus whose metanephric ducts were filled with EGFP signal (Fig. 8C). Of the 264 embryos transferred, four chimeras were born from one surrogate mother (Fig. 9A); two fetuses died on the second day and one was eaten by the surrogate, with another one left for analysis. This one had strong EGFP signals in both the kidneys and the heart (Fig. 9B and C). With the complemented kidneys hosting EGFP+ cells (Fig. 9D), the fetus reached its full-term development; presumably, it was the injected ES cells that successfully compensated for the lost organ during nephrogenesis to support its subsequent embryonic development.

Table 2 Development of blastocyst complemented mouse fetuses at various developmental stages

Full size table

Fig. 6
figure 6

Degenerated mouse fetuses developed in vivo after pronucleus and blastocyst injections. (A) The pregnant uterine horns were dissected from an E13.5 recipient mouse. (B) The upper panel shows the bright-field (BF, left) and EGFP fluorescence (right) images of the same fetus. The lower panel shows the degenerated fetuses (BF: left; EGFP: right). (C) A retarded E15.5 embryo has weak EGFP signal (right) corresponding to its BF image at the left. (D) The degenerated fetal membranes and fetuses at E15.5 (BF: left; EGFP: right). Scale bar: 2 mm

Full size image

Fig. 7
figure 7

Representative mouse fetuses (E13.5, #1-#4) were harvested from the constructs and ES cell injected group (complementation batch). The expression of EGFP fluorescence can be observed in fetuses of EGFP transgenic mice (#1, positive control) and the complemented but degenerated fetus (#4). No positive EGFP signal is observable in fetus #2 and fetus #3. Scale bar: 2 mm

Full size image

Fig. 8
figure 8

Immunohistochemistry sections of mouse fetuses developed to E12.5-E13.5 by embryo transfer after pronucleus and blastocyst injection. (A) An E12.5 mouse fetus with low-level (EGFP fluorescence) tissue and metanephric (white dotted circle) chimerism has no kidney complementation. (B) An E13.5 mouse fetus with a high degree EGFP chimeric signals across different tissues, but low in the metanephric region (white dotted circle), has no complemented kidney observale. (C) An E12.5 mouse fetus exhibits a high degree of EGFP chimeric signals in the metanephric region (white dotted circle). Scale bar: 200 μm

Full size image

Fig. 9
figure 9

Neonatal mouse chimeras were born after double injections and embryo transfer. (A) Four chimeras were born; two of them died within 24 h after. The urinary (B) and the cardiac (C) systems of one neonatal chimera (dead) were analyzed. k: kidney; b: bladder; h: heart. (D) Immunohistochemical sections of kidneys from (B). Scale bar: 200 μm

Full size image

Discussion

In the present study, we have successfully generated exogenous mouse kidneys by CRISPR/Cas9-mediated blastocyst complementation; however, the mouse fetuses failed to survive to adulthood, similar to the previous report4. Usui et al. (2012) targeted Sall1 gene, which is widely expressed in renal progenitor cells and structures derived from cap mesenchyme, including pre-tubular aggregate, renal vesicle, comma- and S-shaped bodies18. During nephrogenesis, Sall1 is essential for initiating the ureteric bud (UB) invasion into metanephric mesenchyme, but some of the Sall1 mutants still have UB invasion and proceed with branching for several rounds5,18. Unlike the approach by Usui et al. (2012), we instead chose Osr1 as the target gene. The expression of Osr1 in intermediate mesoderm is essential for the elongation of the nephric duct and the specification of the metanephric mesenchyme. The idea behind choosing Osr1, upstream to Sall1, was to cover the development of the whole nephric-reproductive system, and it is likely to overcome the difficulty of complicated cell interactions during complementation. In the best scenario, the reproductive system can also be complemented and, presumably, it would have a better chance to produce Osr1 knockout offspring than that of targeting Sall1. Unfortunately, the lethality of Osr1 knockout fetuses was much greater than Sall1 knockouts, as the Sall1 knockouts die within 24 h after birth5,19. The blastocyst complementation result from Usui et al. (2012) showed 37 (26.8%) pups born, and 5 (3.6%) successful kidney complementation out of 138 injected embryos transferred. In the present study, all of the Osr1 knockout mouse fetuses died around E12.5, with 1.5% pups born and 0.4% complemented kidneys, which is also consistent with previous findings10,11. The possible reason for that is that Osr1 knockout also plays other critical roles in development, such as the atrial septum of the heart. Osr1 mutants are likely to lack the primary atrial septum, causing the hypoplastic venous valve and backflow of blood into systemic veins10. Knockout of such a gene can disrupt normal cardiogenesis, although Sall1 knockout embryos could also cause brain defects that interfere with the newborn pup’s suckling ability. Conditional knockout might be one potential solution to reduce the lethality of Osr1 knockout. Also, a functional assay of the exogenous organ is necessary to determine whether the complemented kidney functions normally.

The CRISPR/Cas9 technique is a powerful tool to edit specific genes of interest. Wu et al. (2017) were the first to use CRISPR/Cas9-mediated blastocyst complementation to study pancreatogenesis, cardiogenesis, as well as eyes and forebrain development of embryos, in which most of the knockout blastocysts for exogenous cell complementation were generated by using SCNT or heterozygous breeding15,16,17. Instead of using SCNT, we simplified the procedure into a novel one-step CRISPR/Cas9-mediated gene knockout by using 2 to 3 sgRNAs and demonstrated a satisfactory efficiency of Osr1 knockout ES cells for the complementary development of the organ. While our knockout efficiency is slightly lower than reported in some research with standard protocols20,21, it may be due to a common issue of the microinjection procedure. We used constant flow to inject the Cas9 constructs into the paternal pronucleus, which could lead to variable amount of solution being introduced into each pronucleus. This inconsistency might result in some zygotes not receiving a sufficient or equal amount of the Cas9 constructs, thus reducing the overall knockout rate. Nevertheless, generating host blastocyst, our method is still more efficient than by heterozygous breeding (33.6% versus 25%) and SCNT. We think the efficiency could be higher with an optimized microinjection protocol, such as dual pronuclear injection22, and a quantitative microinjection system to ensure an adequate amount of the Cas9 constructs are injected into the zygotes. Also, with multiple sgRNA injection, it is possible to increase the number of sgRNA constructs for higher efficiency, but its viability also needs to be carefully evaluated due to potential off-target effects or adverse impact on the genomic stability. Although this procedure is highly feasible, like other model systems, the complemented organs need to be further assessed for their functions and the extent of contamination by host cells. Among these unresolved questions, low complementation rates and early death of Osr1 knockout embryos can result in spontaneous resorption of fetuses in the uterus23. This phenomenon occurred after the injection of the constructs, and presumably, the main cause could be due to the knockout of Osr1, even though the spontaneous resorption could also occur in normally developing embryos. Interestingly, the timing of embryo degeneration started one day earlier in those with construct injection only, compared to those followed by the injection of EGFP+ ES cells for complementation. Such early resorption has caused sampling difficulty for genotyping the non-EGFP+ cells, i.e., the host cells, in the complemented organs. Therefore, it appears that using SCNT or heterozygous breeding to generate knockout blastocysts might still be an option in spite of its high cost and time-consuming. The EGFP+ cell-complemented embryos/fetuses produced with this procedure resulted in early lethality and/or low complementation rates.

Besides the difficulties in establishing the complemented organ, there are also concerns about the complemented organs not being “clean” enough, i.e., free from the host cells, for potential transplantation24. Recently, pig organs with α-gal and some other critical gene knockouts (GalSafe) have been successfully transplanted into humans and non-human primates25,26,27,28. The GalSafe pigs have multiple antigen knockouts and human transgenes to minimize the immune rejection and overgrown of the transplanted organ in the human body. Therefore, GalSafe pig organs were found functionally normal without hyperacute rejection in human recipients. In nonhuman primates, the transplanted kidneys can sustain the recipients for up to 2 years27. However, the patients who received humanized porcine organs died from organ failure caused by antigen-mediated rejection, even though the donor pigs had 69 genes edited29,30,31. It suggests that there are still some mechanisms for transplant rejection undiscovered. It is also extremely challenging to edit and maintain the pig strain with numerous modified genes. In comparison, the organs produced by blastocyst complementation are theoretically more immunocompatible, but more difficult to find the right niche for the complement organ and overcome the hurdle of interspecies chimerism32,33. Nevertheless, xenotransplantation of SCNT pig organs to humans is already in clinical trials, whereas organs produced by blastocyst complementation technology are still far from applications, which can serve as an ambitious, long-term goal to meet human organ deficits. In other words, it is potentially possible that further editing of the GalSafe pig cells by the CRISPR/Cas9 technique in combination with blastocyst complementation could mitigate the long-term rejection of the transplants by the recipient.

Materials and methods

Animals

The protocols for animal use and care in this study were approved by the Institutional Animal Care and Use Committee (IACUC) of the National Chung Hsing University, Taiwan (approval number 105–150). All experiments were performed in accordance with the relevant guidelines and regulations, as well as with the ARRIVE guidelines34. Mice (CD1) were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan) and maintained in the vivarium with a 12 h light-dark regime, controlled temperature (20–24 °C), and proper humidity (50–70%). Food and water were accessed ad libitum. The 8-week-old mice (male: 31–33 g; female: 25–27 g) were anesthetized using isoflurane (3.5% for induction and 2–3% for maintenance) during all surgical procedures. Anesthesia depth was monitored by toe-pinch reflex and respiratory rate. For embryo collections and at the end of the experimental procedures, mice were humanely euthanized by carbon dioxide (CO2) overdose in a gradual fill chamber (~ 20%/min). Death was confirmed by cervical dislocation or observation of cessation of respiration and cardiac activity.

Embryo recovery and in vitro culture

CD1 female mice at 8-week of age were administered with 10 IU PMSG (Prospec, HOR-272) by intraperitoneal injection 48 h before mating. On the day of mating, female mice were injected with 10 IU hCG (Sigma, CG10) and were sacrificed at 3.5 dpc. Blastocyst embryos were collected by flushing mouse oviducts and uteri with M2 medium. Harvested blastocysts are cultured in KSOM medium (Millipore, MR-121-D) in an incubator (37 °C) with 5% CO235.

Culture of embryonic stem (ES) cells

The EGFP+ mouse ES cells were generously provided by Dr. Kun-Hsiung Lee from Animal Technology Institute Taiwan (ATIT), Taiwan. The cell line (ESC26) has been proved to be pluripotent36 and possesses the ability of germline transmission37. The protocol for ES cell culture was modified from Czechanski et al. (2014)38. In brief, ESC26 were maintained under 5% CO2 at 37 °C on mitomycin C-inactivated MEF feeder cells (~ 1*105/cm2), which were prepared on gelatin-coated dishes at least one day before seeding ES cells, and then were cultured in 2i/LIF medium containing 77% knockout DMEM, 20% knockout serum replacement, 1% non-essential amino acids, 1% GlutaMax, 1% penicillin-streptomycin, 0.1 mM β-mercaptoethanol, 103 IU/ml mLIF (Millipore, ESG1107), 1 µM PD0325901 (Tocris, 4192) and 3 µM CHIR99021 (Cayman, 13122). With TrypLE, ES cells are passaged every 2 to 4 days before the growing colonies contact neighboring ones.

CRISPR/Cas9 design and genotyping

Functional disruption of the Osr1 gene has been successfully proved by conventional gene knockout strategy10. We utilized the CRISPR/Cas9 gene editing system to target the first coding exon (exon 2) with three sgRNAs that potentially generate different combinations of deletion and/or indels to better achieve the knockout purpose (in embryos). The construct was delivered through microinjection. In brief, the aliquoted sgRNAs and Cas9 protein (SpCas9 2NLS nuclease, Synthego) were premixed (Cas9 protein: 1 µM; sgRNAs: 2µM; 10 µL in total) and incubated at 37 °C for 15 min to form ribonucleoprotein right before microinjection. Different sgRNA premixes were prepared individually for forming RNPs, and then two or three RNPs were mixed for injection. The micropipette (B-100-75-10 for holding and BF-100-78-10 for injection, Sutter) were pulled by the puller (P-97, Sutter), then was cut and modified on the microforge (MF-900, Narishige) right before microinjection. The RNP mixture was loaded into the micropipette of the injection side. The injection micropipette was put on the Sutter Manual Microinjector (Eppendorf), gently hit the holding pipette, then adjusted the pressure to create a continuous flow of the RNP mixture we prepared. We injected the RNP mixture into the paternal pronucleus, and an expansion could be seen when the solution was injected into it. The manipulation time and the flow pressure were optimized to minimize the damage to the embryos.

For Genotyping, the genomic DNA was extracted from individual blastocysts with Allele-in-One Mouse Tail Direct PCR Kit (ABP-RD-2112) for genotyping by using nested PCR. The first PCR used primers 1 and 4 to amplify a 744 bp product (25 cycles), then used primers 2 and 3 to amplify a 287 bp product from the first PCR product (25 cycles). The final PCR products were then transferred to a 96-well plate and analyzed by capillary electrophoresis using a Caliper LabChip GX (LabX). The genotyping of the ES cells used QIAamp® DNA micro kit (QIAGEN, 56304) to extract genomic DNA, then used primer 1 and 5 to amplify a 929 bp product (40 cycles). The products were premixed with loading dye and electrophoresed on a 1.5% agarose gel. The sgRNA sequences and primers are listed in Table 3:

Table 3 Sequences of primer and SgRNAs used

Full size table

Injection of ES cells into blastocyst embryos

Mouse ES cell colonies were disassociated with trypsin-EDTA (Gibco, 15400054). After centrifugation at 1500 rpm for 5 min, single cells were transferred to a 0.2% gelatin-coated dish for 1 h; approximately 10 to 20 ES cells were injected into each E3.5 blastocyst with micromanipulators. The injected blastocysts were then transferred to KSOM medium and incubated at 37 °C for 2 h. Those blastocysts with recovered blastocoels were selected then transfer into the pseudo-pregnant surrogate mice for further experiments39.

Observation of mouse fetuses

The embryo recipients were sacrificed and E12.5 to E15.5 mouse fetuses were collected. These fetuses were fixed in Bouin’s solution (Sigma, HT10132), and then sent to a local company (CIS-BIOTECHNOLOGY CO., LTD.) for paraffin embedding, sectioning, and HE or immunohistochemical staining (EGFP antibody, A-11122, Invitrogen). The sections of mouse fetuses were examined under the microscope (Leica, DM2500) and the images were captured with a CCD camera (LeadView, 2000AIO).

Statistical analysis

All the E12.5 to E15.5 and birth group data presented are at least three biological replicates. Statistical differences between treatment groups were assessed using a two-tailed Student’s t-test of SAS software (version 9.4; SAS Institute, Cary, NC, USA). Any two groups marked without the same alphabetic letters indicate a significant difference (P < 0.05).

Data availability

The data associated with this study are available from the first author and the corresponding author upon reasonable request.

References

  1. Ikada, Y. Challenges in tissue engineering. J. R Soc. Interface. 3, 589–601 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Kobayashi, T. et al. Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell 142, 787–799 (2010).

    Article  CAS  PubMed  Google Scholar 

  3. Matsunari, H. et al. Blastocyst complementation generates exogenic pancreas in vivo in apancreatic cloned pigs. Proc. Natl. Acad. Sci. U S A. 110, 4557–4562 (2013).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  4. Usui, J. et al. Generation of kidney from pluripotent stem cells via blastocyst complementation. Am. J. Pathol. 180, 2417–2426 (2012).

    Article  CAS  PubMed  Google Scholar 

  5. Nishinakamura, R. et al. Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development. Development 128, 3105–3115 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Harrison, S. J., Nishinakamura, R. & Monaghan, A. P. Sall1 regulates mitral cell development and olfactory nerve extension in the developing olfactory bulb. Cereb. Cortex. 18, 1604–1617 (2008).

    Article  PubMed  Google Scholar 

  7. Horster, M. F., Braun, G. S. & Huber, S. M. Embryonic renal epithelia: induction, nephrogenesis, and cell differentiation. Physiol. Rev. 79, 1157–1191 (1999).

    Article  CAS  PubMed  Google Scholar 

  8. Costantini, F. & Shakya, R. GDNF/Ret signaling and the development of the kidney. Bioessays 28, 117–127 (2006).

    Article  CAS  PubMed  Google Scholar 

  9. Xu, P. X. et al. Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nat. Genet. 23, 113–117 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Wang, Q., Lan, Y., Cho, E. S., Maltby, K. M. & Jiang, R. Odd-skipped related 1 (Odd 1) is an essential regulator of heart and urogenital development. Dev. Biol. 288, 582–594 (2005).

    Article  CAS  PubMed  Google Scholar 

  11. James, R. G., Kamei, C. N., Wang, Q., Jiang, R. & Schultheiss, T. M. Odd-skipped related 1 is required for development of the metanephric kidney and regulates formation and differentiation of kidney precursor cells. Development 133, 2995–3004 (2006).

    Article  CAS  PubMed  Google Scholar 

  12. Lin, J. et al. Defects in trophoblast cell lineage account for the impaired in vivo development of cloned embryos generated by somatic nuclear transfer. Cell. Stem Cell. 8, 371–375 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Wu, D. Y. et al. Defective chromatin architectures in embryonic stem cells derived from somatic cell nuclear transfer impair their differentiation potentials. Cell Death Dis. 12, 1085 (2021).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang, X. et al. Epigenetic reprogramming during somatic cell nuclear transfer: recent progress and future directions. Front. Genet. 11, 205 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Wu, J. et al. Interspecies chimerism with mammalian pluripotent stem cells. Cell 168, 473–486e415 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Huang, J. et al. Interspecies blastocyst complementation generates functional rat cell-derived forebrain tissues in mice. bioRxiv, 2023.2004.2013.536774 (2023).

  17. Wu, J. et al. CRISPR-Cas9 mediated one-step disabling of pancreatogenesis in pigs. Sci. Rep. 7, 10487 (2017).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  18. Basta, J. M., Robbins, L., Kiefer, S. M., Dorsett, D. & Rauchman, M. Sall1 balances self-renewal and differentiation of renal progenitor cells. Development 141, 1047–1058 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Nishinakamura, R. & Takasato, M. Essential roles of Sall1 in kidney development. Kidney Int. 68, 1948–1950 (2005).

    Article  CAS  PubMed  Google Scholar 

  20. Yang, H., Wang, H. & Jaenisch, R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. Nat. Protoc. 9, 1956–1968 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Gertsenstein, M. & Nutter, L. M. Production of knockout mouse lines with Cas9. Methods 191, 32–43 (2021).

    Article  CAS  PubMed  Google Scholar 

  22. Abe, T., Inoue, K. I., Furuta, Y. & Kiyonari, H. Pronuclear microinjection during S-Phase increases the efficiency of CRISPR-Cas9-Assisted knockin of large DNA donors in mouse zygotes. Cell. Rep. 31, 107653 (2020).

    Article  CAS  PubMed  Google Scholar 

  23. Drews, B., Landaverde, L. F., Kühl, A. & Drews, U. Spontaneous embryo resorption in the mouse is triggered by embryonic apoptosis followed by rapid removal via maternal sterile purulent inflammation. BMC Dev. Biol. 20, 1 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Freedman, B. S. Hopes and difficulties for blastocyst complementation. Nephron 138, 42–47 (2018).

    Article  CAS  PubMed  Google Scholar 

  25. Porrett, P. M. et al. First clinical-grade Porcine kidney xenotransplant using a human decedent model. Am. J. Transpl. 22, 1037–1053 (2022).

    Article  Google Scholar 

  26. Moazami, N. et al. Pig-to-human heart xenotransplantation in two recently deceased human recipients. Nat. Med. 29, 1989–1997 (2023).

    Article  CAS  PubMed  Google Scholar 

  27. Anand, R. P. et al. Design and testing of a humanized Porcine donor for xenotransplantation. Nature 622, 393–401 (2023).

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mohiuddin, M. M. et al. Progressive genetic modifications of Porcine cardiac xenografts extend survival to 9 months. Xenotransplantation 29, e12744 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Mohiuddin, M. M. et al. Graft dysfunction in compassionate use of genetically engineered pig-to-human cardiac xenotransplantation: a case report. Lancet 402, 397–410 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Schmauch, E. et al. Integrative multi-omics profiling in human decedents receiving pig heart xenografts. Nat. Med. 30, 1448–1460 (2024).

    Article  CAS  PubMed  Google Scholar 

  31. Pan, W. et al. Cellular dynamics in pig-to-human kidney xenotransplantation. Med 5, 1016–1029e1014 (2024).

    Article  CAS  PubMed  Google Scholar 

  32. Sarmah, H. et al. Towards human organ generation using interspecies blastocyst complementation: challenges and perspectives for therapy. Front. Cell. Dev. Biol. 11, 1070560 (2023).

    Article  PubMed  PubMed Central  Google Scholar 

  33. Yamaguchi, T. et al. An interspecies barrier to tetraploid complementation and chimera formation. Sci. Rep. 8, 15289 (2018).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

  34. Percie du Sert. The ARRIVE guidelines 2.0: updated guidelines for reporting animal research. BMC Vet. Res. 16, 242 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  35. Bedzhov, I., Leung, C. Y., Bialecka, M. & Zernicka-Goetz, M. In vitro culture of mouse blastocysts beyond the implantation stages. Nat. Protoc. 9, 2732–2739 (2014).

    Article  CAS  PubMed  Google Scholar 

  36. Lee, K. H., Tu, C. F., Wu, S. C. & Hsu, W. C. Routine generation of green fluorescent chimeric mice with a high percentage of germline transmission by a novel embryonic stem cell line. J. Chin. Soc. Anim. Sci. 32, 143–154 (2003).

    Google Scholar 

  37. Lee, K. H. et al. An alternative simple method for mass production of chimeric embryos by coculturing denuded embryos and embryonic stem cells in Eppendorf vials. Theriogenology 67, 228–237 (2007).

    Article  PubMed  Google Scholar 

  38. Czechanski, A. et al. Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nat. Protoc. 9, 559–574 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wen, D., Saiz, N., Rosenwaks, Z., Hadjantonakis, A. K. & Rafii, S. Completely ES cell-derived mice produced by tetraploid complementation using inner cell mass (ICM) deficient blastocysts. PLoS One. 9, e94730 (2014).

    Article  ADS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank Dr. Kun-Hsiung Lee who provided us the ESC26 as a gift. We are also grateful to the Department of Animal Science, National Chung Hsing University for providing us with the space and equipment for the entire study. Ting-Yu Yeh carried out this research with the funding support in part by the Doctoral Program in Tissue Engineering and Regenerative Medicine of National Chung Hsing University and National Health Research Institutes. This research was funded by the Ministry of Science and Technology, Taiwan (108-2313-B-039-002, 109-2313-B-039-001 and 110-2313-B-039-003-MY2).

Funding

This research was funded by the Ministry of Science and Technology, Taiwan (108-2313-B-039-002, 109-2313-B-039-001 and 110-2313-B-039-003-MY2).

Author information

Author notes

  1. Ting-Yu Yeh and Yu-Jia Huang contributed equally to this work.

Authors and Affiliations

  1. Doctoral Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, Taichung, 402, Taiwan

    Ting-Yu Yeh, Ing-Ming Chiu & Gou-Jen Wang

  2. Neuroscience and Brain Disease Center, China Medical University, Taichung, 404, Taiwan

    Ing-Ming Chiu

  3. Graduate Institute of Biomedical Sciences, China Medical University, Taichung, 404, Taiwan

    Ing-Ming Chiu & Jyh-Cherng Ju

  4. Department of Internal Medicine, The Ohio State University, Columbus, OH, 43210, USA

    Ing-Ming Chiu

  5. Department of Animal Science and Technology, National Taiwan University, Taipei, 106, Taiwan

    Yu-Jia Huang & Shinn-Chih Wu

  6. National Laboratory Animal Center, National Applied Research Laboratories, Taipei, 115, Taiwan

    Chia-Chun Hsieh & Hsiang-Hsuan Sung

  7. Center for Precision Medicine, China Medical University Hospital, Taichung, 404, Taiwan

    Wuh-Liang Hwu & Jyh-Cherng Ju

  8. Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung, 402, Taiwan

    Gou-Jen Wang

  9. Department of Mechanical Engineering, National Chung Hsing University, Taichung, 402, Taiwan

    Gou-Jen Wang

  10. Department of Animal Science, National Chung Hsing University, Taichung, 402, Taiwan

    Jyh-Cherng Ju

Authors

  1. Ting-Yu Yeh
  2. Ing-Ming Chiu
  3. Yu-Jia Huang
  4. Shinn-Chih Wu
  5. Chia-Chun Hsieh
  6. Hsiang-Hsuan Sung
  7. Wuh-Liang Hwu
  8. Gou-Jen Wang
  9. Jyh-Cherng Ju

Contributions

Conceptualization, T.-Y.Y. and J.-C.J.; methodology, T.-Y.Y., H.-H.S., S.-C.W. and J.-C.J.; validation, T.-Y.Y., Y.-J.H., C.-C.H. and W.-L.H.; investigation, T.-Y.Y. and Y.-J.H.; resources, C.-C.H., W.-L.H., I.-M.C., H.-H.S., S.-C.W. and J.-C.J.; data curation, T.-Y.Y. and Y.-J.H.; writing—original draft preparation, T.-Y.Y.; writing—review and editing, H.-H.S. and J.-C.J.; visualization, T.-Y.Y. and Y.-J.H.; supervision, G.-J.W., I.-M.C., S.-C.W. and J.-C.J.; project administration, I.-M.C., S.-C.W. and J.-C.J.; funding acquisition, I.-M.C. and J.-C.J. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Gou-Jen Wang or Jyh-Cherng Ju.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yeh, TY., Chiu, IM., Huang, YJ. et al. Generation of exogenous kidneys via CRISPR/Cas9 mediated blastocyst complementation targeting Osr1 gene in mice. Sci Rep 15, 38648 (2025). https://doi.org/10.1038/s41598-025-22373-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • DOI: https://doi.org/10.1038/s41598-025-22373-9

Keywords

Related Posts