Exploring the Therapeutic Potential of Human Embryonic Stem Cells in Pediatric Neurotrauma

Funding Type: 
SEED Grant
Grant Number: 
RS1-00377
ICOC Funds Committed: 
$0
Disease Focus: 
Spinal Cord Injury
Neurological Disorders
Stem Cell Use: 
Adult Stem Cell
Embryonic Stem Cell
Public Abstract: 
Traumatic brain injury (TBI) is a major cause of death (50,000/yr) and disability. The Centers for Disease Control and Prevention (CDC) has estimated that approximately 1.4-1.5 million Americans survive TBI annually. The economic burden of TBI in the United States has been estimated at approximately $56.3 billion in 1995. In children TBI has lifelong cognitive, physical, psychosocial/ behavioral/ emotional impairments since it interferes with the developing brain. In fact, in children deficits may not be fully revealed until later in life. TBI is among the most frequent pediatric neurological conditions (400,000 emergency department visits/yr; Langlois, 2001), occurring more frequently than cerebral palsy (10,000/yr), global developmental delay (80,000/yr) and epilepsy (30,000/yr). TBI continues to pose a serious health concern and adequate treatment for these injuries is seriously lacking. For example, the majority of TBI patients are discharged without subsequent health-care assistance. Among children aged 0-4 years 91% are unattended clinically post discharge (compare to 66% for all injuries). This is particularly discordant with data showing that at least 15% of TBI patients continue to experience negative consequences 1yr after injury (Guerrero et al, 2000). Optimistically, the neonatal brain may be more amenable than the adult brain to the therapeutic potential of stem cells. Transplantation studies using human embryonic stem cells and other stem cells have demonstrated their ability to facilitate functional recovery after brain and spinal cord injury. The proposed studies will determine the therapeutic capacity of neuron-enriched human embryonic stem cells (hESCs) to attenuate histological damage, normalize brain metabolite profiles, generate functional neural circuits and recover sensorimotor and memory functions following transplantation into postnatal rats subjected to lateral fluid percussion injury (FPI). The lateral FPI model most closely mimics postlesional events associated with TBI in humans (Dietrich et al, 1996). In Aim 1 we will evaluate whether hESCs (previously differentiated into neurons and labeled ferromagnetically) transplanted into the traumatically injured pediatric brain migrate to the site of injury and compensate for lesion area and brain metabolite changes using brain imaging methods. In Aim 2 accelerating rotarod and Morris water maze tests will be used to investigate whether grafted hESCs can ameliorate sensorimotor and cognitive deficits. In Aim 3 we will use immunohistochemical methods to label neural and glial phenotypes and synaptic contacts to determine if grafted hESCs form functional neuronal networks in injured brain. Our findings should eventually help promote the development of novel strategies to alleviate the effects of TBI. The proof of concept obtained in these studies will provide the necessary preliminary data and expertise necessary to be competitive for NIH funding.
Statement of Benefit to California: 
Traumatic brain injury (TBI) is a major cause of death (50,000/yr) and disability. The Centers for Disease Control and Prevention (CDC) has estimated that approximately 1.4-1.5 million Americans survive TBI annually. In contrast, the incidence of breast cancer and HIV/AIDS is 8 and 34 times lower (National Center for Injury Prevention and Control, CDC, http://www.cdc.gov/ncipc/factsheets/tbi.htm). The economic burden of TBI in the United States has been estimated at approximately $56.3 billion in 1995. In addition, TBI imposes enormous losses to individuals, their families, and society that cannot be completely enumerated. TBI is among the most frequent pediatric neurological conditions (400,000 emergency department visits; Langlois, 2001), occurring more frequently than cerebral palsy (10,000/year), global developmental delay (80,000/yr) and epilepsy (30,000/yr). TBI also accounts for significant neurological and neuropsychological morbidity in children. Data from 12 states, including California, indicate that the age-adjusted rate for TBI-related hospitalizations in 2002 was 79.0 per 100,000 population. While this estimate is ~20% lower than annual estimates for 1994-1995 from the National Hospital Discharge Survey (NHDS), TBI continues to leave an estimated 80,000-90,000 persons with long-term disability (Langlois et al, 2004). This apparent decline should be interpreted cautiously as estimates exclude non-residents and brain injuries not treated at hospitals. Most TBI cases are mild and deficits are subtle. In children deficits may not be fully revealed until later in life. TBI, which produces cognitive, physical and psychosocial/ behavioral/ emotional impairments, that often continue to develop for up to months and years after impact, continues to pose a serious health concern and adequate treatment for these injuries is seriously lacking. For example, the majority of TBI patients are discharged without subsequent health-care assistance. Among children aged 0-4 years 91% are unattended clinically post discharge (compare to 66% for all injuries). This is particularly discordant with data showing that at least 15% of TBI patients continue to experience negative consequences 1yr after injury (Guerrero et al, 2000). Optimistically, neonatal brain injury may be more amenable than the adult brain to the therapeutic potential of stem cells (Santner-Nanan et al, 2005). Transplantation studies using non-embryonic stem cells from human fetus, mouse embryonic stem cells have demonstrated their ability to facilitate functional recovery after brain and spinal cord injury. hESCs offer greater ability of pluropotency and can prevent any cross-species consequences. To date a very limited number of studies have explored the ability of hESCs to reverse or protect against detrimental and permanent effects of TBI, especially as measured noninvasively using brain imaging.
Progress Report: 
  • We 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.

© 2013 California Institute for Regenerative Medicine