The liver is the largest internal organ and the center of homeostatic metabolism. Liver-directed cell transplantation is, therefore, an attractive therapeutic option to treat various metabolic disorders as well as liver diseases. Although clinical liver-directed cell transplantation requires multiple cell injections into the portal venous system, a mouse model is lacking which allows us to perform repetitive cell injections into the portal venous system. Here, we propose a surgical model that utilizes the spleen as a subcutaneous injection port. Mouse spleens were translocated under the skin with intact vascular pedicles. Human placental stem cell transplantations were performed one week following this port construction and repeated three times. Cell distribution was analyzed by quantifying human DNA using human Alu-specific primers. About 50% of the transplanted cells were located homogeneously in the liver one hour after the splenic port injection. Fluorescent-labeled cell tracking and antihuman mitochondrion immunohistochemistry studies demonstrated that the cells localized predominantly in small distal portal branches. A similar cell distribution was observed after multiple cell injections. These data confirm that the subcutaneous splenic injection port is suitable for performing repetitive cell transplantation into the portal venous system of mouse models.
Hepatocyte transplantation is one of the promising regenerative approaches for the restoration of or compensation for impaired liver functions [
As stem cell technology has advanced, there has been an increased demand for a suitable rodent model to perform preclinical liver-directed cell transplantation studies. When used clinically for the treatment of human disease, hepatocytes are transplanted multiple times into the portal circulation in order to obtain a therapeutic dose. This is accomplished via a catheter placed in either the intrahepatic portal vein, the middle colic vein, the inferior mesenteric vein, or the patent umbilical vein in the case of neonatal recipients. However, due to size limitations, cell injection routes are limited in rodent models. The size of the mouse mesenteric vein and the difficulty of obtaining hemostasis at this site prohibit its use as an injection site. Although direct liver injection can be used in hairless neonatal mouse models, the direct liver injection approach cannot avoid the possibility of injecting cells into the hepatic venous system, and obtaining hemostasis can be difficult. This makes intrasplenic cell injection the current “gold standard” procedure for cell transplantation in rodent models. Intrasplenic cell injection also minimizes the risk of causing an increase in portal pressure as the spleen can serve as a pressure buffer zone.
Here, we report a surgical procedure to mobilize the mouse spleen into a subcutaneous pocket that was utilized as a subcutaneous injection port for the performance of repeated intraportal venous cell injections. The in vivo distribution of these transplanted cells was subsequently evaluated in this mouse model.
All mice used in this study were bred and euthanized appropriately following the protocols that were approved by the University of Southern California Institutional Animal Care and Use Committee and conducted following the NIH Guide for the Care and Use of Laboratory Animals. Breeder heterozygous pairs of NOD.129(B6)-Prkdcscid Iduatm1Clk mice were obtained from The Jackson Laboratory (#004083), housed under specific-pathogen-free conditions and provided with regular chow (TEKLAD #2018) and sterile/acidified water. PCR-based genotyping was performed with specific primers according to The Jackson Laboratory’s instructions.
One week prior to planned cell injections, the mouse splenic port was prepared by the following technique. Briefly, the hair of the left upper back and trunk area was shaved and chemically removed using depilatory cream. A skin incision was then made over the spleen, and a subcutaneous pocket was then prepared with a straight hemostat (Figure
Construction of the subcutaneous splenic injection port. (a) After hair removal from the surgical area, a subcutaneous pocket was created with a straight hemostat. (b) The spleen was translocated into the subcutaneous pocket. (c) The muscle layer was closed deep to the spleen. (d) Macro view of the translocated spleen. (e) The traced illustration indicates the avascular area of the splenic vascular pedicle. (f) Macro view of the closed muscle layer. (g) The traced illustration depicts the closure of the muscle layer supporting the spleen in the developed subcutaneous pocket. (h) One week following surgery. (i) hAECs were resuspended in 50% trypan blue/PBS solution and transdermally injected into the spleen. (j) One hour after the cell injection, no leakage was observed in the pocket.
Primary human amniotic epithelial cells (hAECs) from five different donors were immortalized using a SV40 Lentiviral vector (pLenti-SV40-T+t, Applied Biological Materials Inc., Richmond, BC, Canada). One line (iAE124) was selected and used for further lentiviral GFP labeling (PL-SIN-EF1a-EGFP).
One week following construction of the subcutaneous splenic injection port, cell injection was performed using the GFP-positive immortalized hAECs (iAE124-GFP) and primary hAECs. A total of 1.5 million cells were suspended in 200
One hour after cell injections were performed, the animals were euthanized and the cell injection site and surrounding tissue were examined for the trypan blue staining (Figure
Cell distribution following intrasplenic cell injection. (a) Illustration of mouse liver lobes and flow of the injected cells via the splenic vein to branches of the portal vein: left lateral lobe (LLL), middle left lobe (MLL), middle right lobe (MRL), right lateral lobe (RLL), and caudate lobe (CL). (b) Phase-contrast image shows the morphology of the immortalized human amniotic epithelial cells (hAECs). (c) The histogram of FACS prior to sorting. (d) Fluorescent image demonstrating the green fluorescent protein- (GFP-) labeled immortalized hAECs after sorting. (e) Cell distribution pie chart. The left chart demonstrates the ratio of human DNA to total DNA from each tissue. The right chart demonstrates the ratio of adjusted human DNA quantity in each liver lobe. (f) A dot plot demonstrating the correlation of the size of each lobe and adjusted quantity of human DNA in each lobe.
In order to detect hAECs in the recipient mouse liver, two cell identification methods, fluorescent-labeled cell tracing (GFP-positive hAEC injection) and antihuman mitochondrion immunohistochemical staining, were used.
Recipient livers were sliced at 5 mm thickness and immersed in 4% paraformaldehyde/PBS fixation buffer overnight at 4°C. The samples were divided in two groups for cryosection and for paraffin embedding/section. The cryosection samples were embedded in an OCT compound and sectioned at 8
Results are expressed as
First, we have established a surgical procedure which involves detachment of the spleen from the stomach and its relocation to the subcutaneous pocket with an intact vascular pedicle. Unlike conventional splenic injections, a relatively large skin incision was required over the upper portion of the spleen. To access the gastrosplenic ligament, the greater curvature of the stomach was grasped and used for manipulation. To test the feasibility of this surgical procedure for future usage with disease mouse models, we used semi-immunodeficient Idua knockout mice as the recipients. Despite using this relatively fragile disease model mouse, there was no mortality associated with the procedure. One complication included a failure of skin closure due to the insufficient size of the subcutaneous pocket, suggesting that a generous subcutaneous pocket should be developed in order to have a sufficient space to accommodate the spleen (Figure
Primary human amniotic epithelial cells (hAECs) from five different donors were immortalized using a SV40 Lentiviral vector (pLenti-SV40-T+t, Applied Biological Materials Inc., Richmond, BC, Canada). The established immortalized hAEC lines were morphologically evaluated (Figure
To evaluate the in vivo cell distribution following splenic reservoir cell injections, the presence of human DNA was quantified by qPCR using human Alu sequence-specific primers [
We further investigated the detailed intrahepatic cell distribution by dissecting the liver lobes. The quantity of human DNA in each liver lobe was estimated, and the intrahepatic cell distribution ratio was calculated (Figure
GFP-positive cell tracing and antihuman mitochondrion immunohistochemistry demonstrated that the cells were heterogeneously located within each of the lobes. The GFP-positive cells were found predominantly in small distal portal branches at an average distance from the liver surface of
GFP-labeled immortalized hAEC injection. (a) The low magnification fluorescent image (×10) of a recipient liver one hour after cell injection. (b) The high magnification fluorescent image revealed GFP-positive cells in the liver parenchyma (red arrow). DAPI (blue) and Alexa Fluor 594 Phalloidin (red) were used as counterstains to visualize the nuclear and liver structures, respectively. (c) Antihuman mitochondrion immunohistochemistry demonstrates cell distribution and microemboli in the mouse portal vein. (d) Antihuman mitochondrion immunohistochemistry demonstrates human cells in the mouse liver parenchyma.
The function of the subcutaneous splenic cell injection port was confirmed after multiple hAEC transplantations by injecting GFP-positive hAECs. The subcutaneous spleen was clearly visible under the skin and palpable one month after the surgery. Following three injections of non-GFP-labeled hAECs at one week intervals, GFP-positive immortalized hAECs were injected (Figure
Multiple human amniotic epithelial cell injections. (a) The schematic shows an overview of the multiple cell injection study design. One week before the first cell transplantation, the subcutaneous cell injection port construction surgery was completed. A total of 1.5 million hAECs were transplanted every week for four times. The final cell injection was performed with GFP-labeled hAECs to confirm the function of the splenic injection port. (b) The low magnification fluorescent image (×10) of the recipient liver one hour after the final GFP-positive cell injection. The yellow arrows indicate the cells located in the area of small distal portal branches. (c) The cell distribution and migration are similar to those of the first cell transplantation after serial cell injections. DAPI (blue) and Alexa Fluor 594 Phalloidin (red) were used as counterstains to visualize nuclear and liver structures, respectively.
Repeated cell injections are often required to transplant a sufficient number of cells for disease phenotype improvement. Clinical hepatocyte transplantation studies indicate that about 100 million cells per kg body weight are required to replace the estimated 5-20% of the patient’s missing enzyme function, necessary for phenotype stabilization/improvement [
In this study, we investigated the in vivo and intrahepatic cell distribution. Although one lung sample contained detectable human DNA, in most of the cases, the injected cells were retained in the spleen or the liver. The intrahepatic cell distribution was correlated with the size of each lobe. This data further supports the previous study using fluorescent beads which demonstrated that portal vein flow is evenly distributed to each lobe [
This model will be useful to optimize various preclinical therapeutic conditions using different disease model mice. We have reported the therapeutic efficacy of hAEC by percutaneous direct liver injection into a neonatal mouse model of mucopolysaccharidosis I [
We have established a mouse model which allows for the minimally invasive performance of multiple cell injections. The cell distribution analyses indicated homogeneous cell distribution between the liver lobes, and heterogeneous distribution within the liver lobes. The subcutaneous splenic cell injection port was functional after multiple cell injections. This model will be useful to simulate clinical hepatocyte transplantation and facilitate preclinical studies using stem cell-derived hepatic cell transplantation studies.
Green fluorescent protein
Fluorescence-activated cell sorting
Human amniotic epithelial cell
Left lateral lobe
Middle left lobe
Middle right lobe
Right lateral lobe
Caudate lobe
Optimal cutting temperature
Phosphate-buffered saline
4
Polymerase chain reaction
Tissue factors.
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
The authors appreciate Dr. Tin Aung Than, Dr. Sanda Win, and Dr. Neil Kaplowitz for providing reagents for cell immortalization. We acknowledge the Analytical/Instrumentation Core of the University of Southern California Research Center for Liver Diseases (P30DK048522) for the use of various instruments. This work was partially supported by California Institute for Regenerative Medicine (CIRM) grant TR3-05488 (TM).