The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique
The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.
1. Preparation of Injection Pipettes
2. in Utero Transplantation
3. Representative Results:
Survival of the injected fetuses to term delivery is the main limiting factor to achieving success with this technique. Depending on the material injected and the strain of mouse being used, survival rates can vary. In general, injections of hematopoietic cells into wild-type mice at E14 should result in at least 50% live-born pups. Higher rates of survival are possible depending on both the technical aspects of the injection as well as the characteristics of the mice being injected.
Minimizing trauma to the uterus and amniotic membranes is the most important technical aspect of this protocol. Sharp, small caliber pipettes will result in minimal uterine trauma during the injection. We do not recommend using standard injection needles as the caliber of pre-fabricated needles is too large and would result in a large hole in the uterus. Hand crafted glass injection pipettes are the only small caliber needles that can be utilized for in utero injections. Careful and meticulous surgical technique, including gentle handling of the uterus and a short anesthetic is also crucial for optimal results.
Specific characteristics of the recipient mice such as the genetic background, gestational age, and litter size can also affect survival. Certain strains of mice are more susceptible to preterm labor and pregnancy loss depending on their genetic background.15 Transgenic animals with muscular or neurodegenerative defects can have an impaired ability to deliver fetuses vaginally after undergoing a midline laparotomy.8 These pregnant females may require delivery by cesarean section. We have also found that the gestational age at the time of injection can impact viability. Fetuses that are younger than embryonic day 12 have lower rates of survival than older fetuses. Finally, we have found that large litter sizes (>10 fetuses) tend to have higher rates of fetal demise after injection. Attention to both the technical aspects of this technique and the specific characteristics of the mice being injected can maximize survival of the injected fetuses.
When these methods are performed correctly, one can expect that all fetuses are exposed to the injected material. Similar to the postnatal setting, however, the successful delivery of cells or viruses in utero does not always result in donor cell engraftment or gene expression, respectively. The engraftment of stem cells, for example, is dependent on several factors such as the dose and source of the transplanted cells. Similarly, the success of viral transduction is, in part, determined by the type of viral vector used. One must understand the numerous factors that impact pup survival, cellular engraftment, and viral transduction to achieve success with this protocol.
Figure 1. Diagram depicting proper sharpening of the injection pipettes (Step 1.4)
Over 50 years ago, Billingham, Brent, and Medawar used in utero transplantation in mice to induce immune tolerance to foreign proteins.16 Since that time, several variations of this technique have been used to address questions in immunology and stem cell biology.
The protocol detailed here is one of the most accessible methods for IUT. The fetal liver offers an easily visualized target and provides access to the systemic circulation via the portal and hepatic veins. However, several modifications have been described in order to optimize the timing and location of the desired injection. For example, injections in fetuses younger than E13.5 can be more difficult because the uterus is not transparent. If this approach is necessary to study earlier events in development, ultrasound-guided injections may be used to target particular tissues.17 Ultrasound guidance is also useful to direct the injection into particular organs, such as the heart or lungs. Intravenous injections into the vitelline vein18,19 offer the advantage of delivering cells directly into the circulation and may allow for the delivery of larger volumes of cells.5 We have intentionally described a general approach to performing IUT in mice so that the reader has the necessary foundation to achieve success with these injections and can tailor these methods to the specific application required.
Despite its initial use to study immune tolerance over 50 years ago, there are still important unanswered questions for which IUT will be instructive. The ability to induce tolerance to specific foreign antigens by their introduction into the fetal environment has been demonstrated in mice.4,6,7 While the mouse fetal immune system develops later than that of larger animals and may impact this finding, the precise mechanisms by which fetal tolerance occurs in mice need to be studied further. Such experiments may elucidate ways to improve tolerance induction in humans. Even in mice, factors such as the availability of hematopoietic niches and the competitive advantage of host cells continue to limit the success of stem cell engraftment.3 Current strategies to further investigate this include manipulating specific mechanisms of the host immune response and optimizing the route, timing, and dose of stem cell delivery. IUT into early gestation mice provides an ideal model in which to study stem cell engraftment and differentiation.
IUT is a powerful tool to understand fundamental issues in the fields of stem cell and developmental biology. Defining the mechanisms which lead to fetal tolerance to antigens introduced in utero will have major implications for the field of stem cell transplantation. In addition, successful delivery of viral vectors may provide a treatment for single gene disorders. The availability of numerous transgenic and mutant mouse strains allows for investigation into the role specific genes play to alter the expected phenotype. The mouse model of IUT will undoubtedly advance these fields and bring us closer to realizing their full clinical potential for treating patients with congenital anomalies.
The authors have nothing to disclose.
We would like to acknowledge our funding sources: The California Institute for Regenerative Medicine Clinical Fellow Training Grant (AN), National Science Foundation (MW), Irene Perstein Award (TCM), American College of Surgeons (TCM), American Pediatric Surgical Association (TCM), and the March of Dimes (TCM).
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
Pipettes | Kimble | 71900-100 | ||
Pipette puller | Sutter Instruments Company | Model P-30 | ||
Microinjector | Narishige | IM-300 | ||
Pipette sharpener | Sutter Instruments Company | Model BV-10 |