Department of Surgery and Cancer

Dr Pascale Guillot

Fetal Stem Cell Therapy

Our group focuses on the biology of human fetal stem cells, with the aim of, firstly, understanding the mechanisms mediating their tissue repair capacity in vivo and, secondly, using this understanding to improve cell therapy paradigms to treat human genetic diseases.

Background

Fetal stem from amniotic fluid and placenta.
Stem cells have been proposed as vehicles for gene or protein delivery because of their ability to expand, differentiate and repopulate a host in vivo. Stem cells transplanted early in life are healthy cellular building blocks that contribute to developing organs and that are stored in the marrow to repair organs or tissues later in life when injury occurs.

Human fetal stem cells have advantageous biological characteristics compared to adult stem cells, i.e. they are smaller in size, express pluripotency markers, grow four times faster, senesce later, are more adherent, less immunogenic, have longer telomeres and differentiate more readily than adult stem cells.
We study the origins and properties of fetal stem cells from amniotic fluid and placenta membrane isolated at various stages of pregnancy.

Fetal stem cell therapy in fetal medicine and paediatrics.
Fetal stem cells can repair bone and kidney.

Bone:
We have transplanted fetal stem cells in utero in dominant negative a mouse model of Osteogenesis Imperfecta (oim mice) in two independent studies. In both studies, we found a two third reduction in long bone fracture rate, and presence of donor cells in bones. Our latest study provided insight into the mechanisms leading from donor cell infusion to reduction of fractures, showing that donor cells produced the protein ColIα2 orignally missing in oim mice, which contributed to increase bone formation, decrease tibial growth plate height (Figure 1), and modify bone matrix composition, increasing long bones plasticity and reducing their brittleness, which led to stronger and straighter bones.

 

Figure 1: Intrauterine transplantation (IUT) of human fetal MSC decreases tibial growth plate height in 4, 8 and 12 weeks old oim mice . Figure 1: Intrauterine transplantation (IUT) of human fetal MSC decreases tibial growth plate height in 4, 8 and 12 weeks old oim mice .

 

Kidney:
Oim mice have a deletion in the alpha 2 chain of procollagen type I gene resulting in the synthesis of abnormal α1(I)3 homotrimers which replace normal α1(I)2α2(I)1 heterotrimers and a glomerulopathy characterized by abnormal collagen deposition in the glomeruli, which increases in frequency and severity with postnatal age. Intrauterine transplantation of human MSC from first trimester fetal blood led postnatally to a reduction of abnormal homotrimeric collagen type I deposition in the glomeruli of 4-12 week old col1α2-deficient mice (Figure 2).  Using bioluminescence imaging, in-situ hybridization and immunohistochemistry in transplanted col1α2-deficient mice, we showed that the damaged kidneys preferentially recruited donor cells in glomeruli, around mesangial cells (Figure 3).

 

Figure 2: Decrease in the proportion of renal glomeruli containing collagen accumulation (stained in red with picrosirius red) in 8 weeks old mice following intrauterine transplantation of human fetal MSC.

Figure 2: Decrease in the proportion of renal glomeruli containing collagen accumulation (stained in red with picrosirius red) in 8 weeks old mice following intrauterine transplantation of human fetal MSC.

 

 

Figure 3: Localisation of donor cells in renal glomeruli by in situ hybridization using a human pan-centromeric probe (A-B) and immunohistochemistry using human specific anti-vimentin visualized by immunofluorescence (C) or light microscopy (D-F).

Figure 3: Localisation of donor cells in renal glomeruli by in situ hybridization using a human pan-centromeric probe (A-B) and immunohistochemistry using human specific anti-vimentin visualized by immunofluorescence (C) or light microscopy (D-F).

 

Aims

  1. To test the ability of human fetal stem cells to differentiate down the podocyte lineage to prevent and repair glomerulopathy in a mouse model of Alport Syndrome.
  2. To analyze the pathways involved in human fetal stem cell migration to bones in a mouse model of Osteogenesis Imperfecta (oim) to increase donor cell uptake and improve the therapeutic benefits of prenatal fetal cell therapy for OI.

Future Research Plans

1.      Podocyte differentiation and kidney repair capacity of human fetal stem cells.

Inherited nephropathies such as Alport syndrome can lead to renal failure and death. We have previously shown that human fetal stem cells transplanted in utero in a mouse suffering from abnormal collagen type I accumulation in renal glomeruli led to a decrease in a glomerulopathy and donor cells present in glomeruli, providing proof of principle for the kidney repair capacity of human fetal stem cells. We now use cell and molecular biology techniques, bioluminescence imaging, ex vivo and in vivo methods to investigate the ability of human fetal stem cells from amniotic fluid and placenta transplanted in utero and/or postnatally to engraft in kidneys in a murine model of Alport syndrome(Col4α3–/– mice model), differentiate into functional podocyte and repair basement membrane collagen defects and prevent or reverse kidney pathology.

 2.      Analysis of the pathways involved in human fetal stem cell migration to bones in a mouse model of Osteogenesis Imperfecta (oim) to increase donor cell uptake and improve the therapeutic benefits of prenatal fetal cell therapy for OI.

We have shown that human fetal stem cells transplanted intraperitoneally in utero in oim homozygous mice, engrafted in all organs, including brain, but preferentially homed to bones, where they were almost exclusively found at site of bone formation and around callus formation, where they produced the protein missing in oim mice. However, when transplanted in a wild type fetus, donor cells did not preferentially homed to bones, indicating the tissue injury is a prerequisite for donor cell recruitment. Albeit engraftment levels remaining low in oim bones, this was associated with an important reduction in long bones fracture rate. This indicates that fetal stem cells transplanted early in life during organ development and before irreversible damage are capable to contribute to bone formation and prevent fracture occurrence as well as being recruited de novo from the bone marrow to repair fractures. We use the oim model, cell biology techniques, chemotaxis assays and in vivo methods to study how donor cells home to developing oim bones and sites of fracture to improve cell uptake in bone and bone marrow, focusing on the SDF-1/CXCR4 -Tnfa axis, in order to develop therapy paradigms as an alternative for OI treatment.

References

  1. Guillot PV, Abass O, Bassett DJH, Shefelbine SJ, Bou-Gharios G, Chan J, Kurata H, Williams G, Polak J, Fisk NM. (2008) Intrauterine transplantation of human fetal mesenchymal stem cells reduces bone pathology in osteogenesis imperfecta mice. Blood 111(3):1717-25.
  2. Guillot PV, Cook TH, Pusey CD, Fisk NM, Bou-Gharios G. (2008) Transplantation of human fetal mesenchymal stem cells improves glomerulopathy in experimental osteogenesis imperfecta. Journal of Pathology, 214(5)627-36.
  3. Guillot PV, De Bari C, Dell’Accio F, Kurata H. Polak J, Fisk NM. (2008) Comparative osteogenic transcription profiling of various fetal and adult mesenchymal stem cell sources. Differentiation, 76(9):946-57.
  4. De Bari C, Guillot PV, Fisk NM, Khan IM, Archer CW, Jones EA, McGonagle D, Luyten FP, Pitzalis C, Dell'Accio F. (2008) A biomarker-based mathematical model to predict bone-forming potency of human synovial and periosteal mesenchymal stem cells. Arthritis and Rheumatism, 58(1):240-250.
  5. Guillot PV, Cui W, Fisk NM, Polak DJ. (2007) Stem cell differentiation and expansion for clinical applications of tissue engineering. J Cell Mol Med. 11(5):935-44.
  6. Kurata H, Guillot PV, Chan J, Fisk NM. (2007) Osterix induces osteogenic gene expression but not differentiation in primary human fetal mesenchymal stem cells. Tissue Engineering, 13:1513-1523.
  7. Guillot PV, Gotherstrom AC, Chan J, Kurata H, Fisk NM. (2007) Human first trimester fetal mesenchymal stem cells (MSC) express pluripotency markers, grow faster and have longer telomeres compared to adult MSC. Stem Cells 25(3):646-654.
  8. Chan J, Waddington SN, O’Donoghue K, Kurata H, Guillot PV, Gotherstrom C, Themis M, Morgan JE, Fisk NM. (2007) Widespread distribution and muscle differentiation of human fetal mesenchymal stem cells after intrauterine transplantation in dystrophic mdx mouse. Stem Cells 25(2): 875-884.
  9. Guillot PV, Cui W, Fisk N, Polak J. (2007) Stem cell differentiation and expansion for clinical applications of tissue engineering. Journal of Cellular and Molecular Medicine 11(5):935-944.
  10. Guillot PV, O’Donoghue K, Fisk NM. (2006) Fetal stem cells: betwixt and between. In:  Seminars in Reproductive Medicine, Stem Cells in Reproductive Medicine, B Carr, J Itskovitz-Eldor (eds), 24(5):340-347.
  11. Gotherstrom C, Guillot PV, Fisk NM. (2006) Fetal mesenchymal stem cells are more primitive than adult mesenchymal stem cells. In: Stem Cell Repair and Regeneration, 2nd ed. NA Habib, MY Gordon, N Levicar, L Jiao, G Thomas-Black (eds). London: Imperial College Press.


Team members
Pascale V Guillot (Head of Research Group)
Dafni Moschidou (KRUK)

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