Statement of Benefit to California:
Year 1We have shown that fetal human central nervous system derived stem cells (HuCNS-SC) transplanted into a mouse model of spinal cord injury (SCI) improve behavioral recovery. Transplanted human cells differentiated into myelinating oligodendrocytes and synapse forming neurons. These data suggest that efficacy is dependent upon successful cell engraftment and appropriate cell fate. The strain of mice (NOD-scid mice) are immunodeficient, which allows transplanted human cell populations to engraft and promote behavioral recovery in the absence of confounds due to a rejection response and allows us to avoid using immunosuppressant drugs. Clinically, however, it is clear that transplantation of therapeutic human cell populations will require administration of immunosuppressants (IS) such as CsA, FK506, or Rapamycin. These immunosuppressants work by altering signaling pathways which are also present within stem cells. Hence, in addition to promoting engraftment, IS have the potential to affect stem cell proliferation and/or differentiation. In Aim 1A, we tested this hypothesis in a cell culture model and found that HuCNS-SC fate and proliferation were altered by exposure to different IS. CsA and FK506 decreased the number of astrocytes in culture compared to control conditions, while Rapamyin increased the number of astrocytes. All three IS increased the number of ß-tubulin III positive neuron-like cells.
In Aim 1B, we tested whether cells of the inflammatory system (neutrophils and macrophages) could also directly influence stem cell proliferation and fate. To test this possibility, we exposed either fetal or embryonic neural stem cells to cell culture media from co-cultures of neutrophils or macrophages. We found that neutrophil-mediated release of inflammatory proteins promotes astrocyte differentiation of fetal derived neural stem cells but not embryonic derived neural stem cells. One way inflammatory cells might be working is via oxidative stress (e.g. hydrogen peroxide). Interestingly, excess hydrogen peroxide promoted more extensive cell death of embryonic derived versus fetal fetal derived neural stem cells, suggesting an intrinsic difference in the vulnerably of these two cell populations to oxidative stress. Conditioned media from neutrophils was found to reduce proliferation in fetal neural stem cells but not embryonic derived neural stem cells. In addition, we found neutrophil conditioned media promotes human fetal NSC astrocytic fate and migration towards sites of injury epicenter in an animal model of spinal cord injury; followup cell culture experiments enabled us to determine that neutrophil synthesized complement proteins were having a direct effect on stem cell fate and migration, resulting in a patent filing. These data demonstrate that fetal NSCs and ES-NSCs are very different by nature and nurture.
In Aim 2, we evaluated the hypothesis that IS could alter stem cell proliferation and/or fate in vivo, independent of rejection from the recipient’s immune system. HuCNS-SC were transplanted into NOD-scid mice, which have no immune system and hence cannot mount an immune response to the foreign cells. These animals received different immunosuppressants (CsA, FK506, Rapamycin, or vehicle) daily after transplantation until sacrifice 13 weeks later to determine if the total number of surviving human cells, or the end cell fate of the transplanted cells would be altered due to exposure to IS drugs compared to the vehicle control group. Behavioral recovery was assessed via open-field walking assessment, horizontal ladder beam testing, and video based “CatWalk” gait analysis. IS administration did not affect behavioral recovery by any of these measures compared to HuCNS-SC transplanted animals that received vehicle as an IS. Spinal cords were dissected, sectioned, and immunostained using human-specific markers in conjunction with cell lineage/fate and proliferation markers. Cell engraftment, proliferation, and fate were quantified using unbiased methods. The average number of engrafted human cells in uninjured animals was 319,700 vs 214,900 in vehicle treated injured controls. Human cell engraftment in any IS group was not significantly different than vehicle injured controls. Interestingly, 67% of human cells differentiated into Olig2+ oligodendrocyte-like cells in the uninjured controls, while 45% were Olig2 positive in vehicle treated injured controls. IS treatment did not alter Olig2 cell numbers in injured animals. 9% of human cells differentiated into GFAP positive astrocyte-like cells in the uninjured controls, compared with 9% in vehicle treated injured controls. IS treatment did not alter GFAP cell numbers in injured animals. Quantification of proliferation and other lineage markers is ongoing. The important finding thus far is that when administered to whole animals with a human stem cell transplant, a range of immunosuppressant drugs does not appear to significantly alter stem cell fate.