| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 9, 2006 as doi:10.1096/fj.05-4684fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,#,1,
,||,#,,,||,¶,#
* Center of Excellence for Aging and Brain Repair, Departments of
Neurosurgery,
Physiology and Biophysics,
Anatomy,
|| Pharmacology and Therapeutics,
¶ Psychiatry,
# Pathology, and
** Division of Comparative Medicine, University of South Florida,
College of Medicine, Tampa, Florida, USA;
Saneron CCEL Therapeutics, Inc., Tampa, Florida, USA; and
All Children’s Hospital, St. Petersburg, Florida, USA
1Correspondence: Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, College of Medicine, 12901 Bruce B. Downs Blvd., MDC 78, Tampa, FL 33612, USA. E-mail: sgarbuzo{at}hsc.usf.edu
SPECIFIC AIM
Numerous data support passage of maternal cells into the fetus during pregnancy in human and animal models, but the functional benefits of maternal microchimerism in utero are unknown. The current study took advantage of this route for prenatal delivery of 
-N-acetylglucosaminidase (Naglu) enzyme into the enzyme-deficient mouse model of Sanfilippo syndrome type B (MPS III B). Enzymatically sufficient mononuclear cells from human umbilical cord blood (MNC hUCB) were administered i.v. into heterozygote females modeling MPS III B on the 5th day of pregnancy during blastocyst implantation.
The aim of this study was to determine whether administered MNC hUCB cells in the pregnant mouse modeling MPS III B migrated to the fetus and corrected the Naglu enzyme levels in the offspring.
PRINCIPAL FINDINGS
1. Administered MNC hUCB cells transmigrated and diffused into the embryos (E12.5)
Seven days after i.v. administration of MNC hUCB cells, embryos/placentas (N=11) from randomly selected treated heterozygote females were analyzed for the presence of transmigrated cells using a specific marker for anti-human nuclei (HuNu). The embryos/placentas (N=8) from media-injected heterozygote female and C57BL/6J female served as controls. Morphological analysis of embryos/placentas was also performed. Examination revealed transmigration of MNC hUCB cells in many areas of the embryos (Fig. 1
). Mainly, the cells were present in the primitive ectomeninx (Fig. 1A
), the loosely packed cephalic mesenchyme (Fig. 1B, C
), the choroid plexus extending into the lateral ventricle (Fig. 1D
), and the choroid plexus extending from the roof of fourth ventricle (Fig. 1E
). Cells were found in areas around the dorsal root ganglion (Fig. 1G
) and the cartilage primordium of the vertebral body (Fig. 1H)
.
|
2. Transmigrated cells expressed CD34 and CD117 antigens
Many cells were identified in the embryo’s liver (Fig. 1F
), and some were CD34 positive (Fig. 1I
). Expression of CD117 was found in some cells in marginal or mantle layers of the spinal cord (Fig. 1J, L
). A cell attached to the ventricle wall displayed epithelial-like cell morphology (Fig. 1M
). No morphological abnormalities were found in fetuses from mothers receiving MNC hUCB cells; their developmental features at this stage (E12.5) were similar to fetuses from media-injected heterozygote or C57BL/6J females.
3. Transmigrated cells were found in maternal and embryonic parts of placentas
Placentas from both treated heterozygote and C57BL/6J females appeared structurally normal, but placental thickness in heterozygote females was significantly reduced compared with control C57BL/6J mice. This difference was due to decreased thickness of the decidua (the maternal part) in the heterozygote females. The chorionic plate in these mice was thinner. Structural characteristics of the embryonically derived labyrinth, spongiotrophoblast, and giant cell trophoblast of heterozygote females were within normal limits. The labyrinth is comprised mainly of embryonal labyrinth trophoblasts, embryonal endothelium-forming blood vessels, and maternal blood cells. Immunohistochemical staining for MNC hUCB cells demonstrated their presence in the deciduas of these heterozygote-treated females. Some cells were observed in the spongiotrophoblast layer, labyrinth, and the chorionic plate.
For additional identification of MNC hUCB cells, Western immunoblot assay was used on embryos and placentas of the MNC hUCB cell-administered, media-injected, and control C57BL/6J mice. There was an intense band (
70 kDa) derived from embryos prenatally receiving MNC hUCB cells. This band was positive for mouse anti-human nuclei monoclonal antibody, similar to that in the positive control cell sample. However, specific protein bands in placentas of treated embryos were not detected and results were similar to those from placentas of control mice.
4. Transmigrated cells corrected Naglu enzyme activity in all embryos
Naglu enzyme assay was performed in all fetuses (N=5, E12.5) from one randomly chosen litter receiving MNC hUCB cells. Naglu enzyme activity was similar in all embryos, ranging from 1.203–1.355 nmol/h/protein, similar to their parents. Enzyme levels detected in heterozygote parents at 2 months of age 1 wk before cell administration were: female, 1.035 nmol/h/protein; male, 1.369 nmol/h/protein. Undeveloped or dead embryos were not found.
5. Administered MNC hUCB cells were extensively distributed in the organs and the blood of heterozygote mothers 1 wk after transplantation
The MNC hUCB cells were present in the brains and abdominal organs in females 1 wk after receiving cells. Cells identified by human nuclei specific antigen expression were found inside and outside the CNS. MNC hUCB cells were identified in the brain (cerebral cortex, hippocampus, choroid plexus, and striatum) and organs (heart, lung, kidney, and spleen). Although most grafted cells were found in the blood vessels of the aforementioned organs, some were observed in the parenchyma.
CONCLUSIONS AND SIGNIFICANCE
Significant evidence supports maternal cells passing into the fetus during pregnancy in both human and animal models. However, functional advantages of maternal microchimerism in utero are still unclear. It is possible that the maternal cells which prenatally transfer into the human fetus as early as the 13th wk of gestation are involved in developing the fetal immune system. Recently, maternal cells were identified in major organs of neonates with lupus syndrome, an autoimmune disease that develops in utero. Most of these maternal cells in the heart expressed a specific marker for cardiac myocytes and just a few cells expressed CD45. Thus, differentiated-tissue-specific maternal microchimerism occurring in neonates points to cellular plasticity of maternal cells within the fetus. It was suggested that "maternal cells could contribute to a secondary process of tissue repair." However, pluropotentiality of the hematopoietic maternal cell needs future investigation.
To our knowledge, this is the first report demonstrating that enzymatically sufficient MNC hUCB cells xenotransplanted into the blood circulation of a mouse modeling MPS III B at the 5th gestation day transmigrate to the developing embryos. Although the migration mechanism is unknown, a possible explanation is that it occurs due to immunological "recognition" of the immune immature hUCB cells. Another possible migratory mechanism is the developing immunotolerance between human and mouse cells, since post-transplant pregnant mice were immunosuppresed with cyclosporine. Mouse E12.5 is an embryonic stage when the cephalic neural tube is already differentiated and extensive fetal capillary formation is seen in the fully formed placenta. Detection of human cell chimerism at this stage in regions of the embryos and placentas from heterozygote mice receiving MNC hUCB cells may indicate the involvement of these transmigrated cells in embryonic development.
Alternatively, MNC hUCB cells delivered into the maternal blood might reach the embryo passively due to placental damage. Ultrastrutural placental abnormalities have been noted as early as 10 wk gestation in various inherited metabolic disorders. In Hurler’s disease (MPS I), stromal cells of the placenta are vacuolated, while collagen fiber disorientation and syncytial vacuolation have been seen in placentas from Sanfilippo syndrome, particularly in a case of MPS III A. In the current study we showed that placental thickness in heterozygote females was significantly reduced compared with C57BL/6J mice, the apparent result of a smaller decidua (the maternal part) and thinner chorionic plate (the embryonic part).
It has been shown that the blood circulation through the mouse placenta is similar to that of humans. Many intervillous channels are tortuous in humans and similar to the trophoblast-lined sinusoids of the mouse. Moreover, the important role of trophoblast giant cells in materno-fetal vascular interaction has been demonstrated. However, the region bordering the maternal surface of the fetal placenta/labyrinth (basal plate in human and junction zone in mice) does not contain fetal blood, but is traversed by maternal blood channels lined by zygote-derived trophoblast cells, through which maternal blood flows into and out of the fetal placenta/labyrinth. Thus, it is likely that transmigration of MNC hUCB cells from the mother into the embryo may occur through spaces lined by embryonic trophoblasts in the placenta.
Assuming simple Mendelian inheritance, the mating of heterozygous (+/–) parents would be expected to produce a ratio of 25% +/+ (wild-type):50% +/– (heterozygote):25% –/– (homozygous) offspring with distinct level of Naglu enzyme activity (normal, half normal, and none, respectively). As we reported earlier, there were no age-related differences in enzyme level among these phenotypic groups of mice. Heterozygote mice contain enzyme from 1.53 to 2.18 nmol/h/protein and wild-type mice from 3.91 to 4.61 nmol/h/protein. When we detected enzyme activity in the embryos (E12.5) 1 wk after delivery of MNC hUCB cells into the pregnant mother, enzyme levels were similar in all embryos, ranging from 1.203–1.355 nmol/h/protein, and similar to their parents. Enzyme activity was not detected prenatally in control untreated heterozygote mice; however, newborn or postnatal mice as early as 10 days of age showed the 3 distinct Naglu levels. It is possible that one mechanism of this prenatal correction of enzyme levels resulted from administration of enzymatically sufficient MNC hUCB cells, which contain and may release Naglu enzyme into the maternal blood circulation, allowing passage of the enzyme to embryos. Our current findings support an alternative mechanism, transmigration of the MNC hUCB cells through the placenta into embryos, as shown by schematic diagram (Fig. 2
).
|
In principle, prenatal delivery of deficient Naglu enzyme by transplantation of enzymatically sufficient cells into the blood circulation of the mother is possible and may have therapeutic potential in treatment of MPS III B. Prenatal treatment may present a significant opportunity for new biotechnologies to treat many inherited disorders.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4684fje;
This article has been cited by other articles:
|
|
 |
|
  C.-P. Chen, M.-Y. Lee, J.-P. Huang, J. D. Aplin, Y.-H. Wu, C.-S. Hu, P.-C. Chen, H. Li, S.-M. Hwang, S.-H. Liu, et al. Trafficking of Multipotent Mesenchymal Stromal Cells from Maternal Circulation Through the Placenta Involves Vascular Endothelial Growth Factor Receptor-1 and Integrins Stem Cells, February 1, 2008; 26(2): 550 - 561. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
