Back

Lee J-P, Jeyakumar M, Gonzalez R, Takahashi H, Lee P-J, Baek RC, Clark D, Rose H, Fu G, Clarke J, McKercher S, Meerloo J, Muller F-J, Park KI, Butters TD, Dwek RA, Schwartz P, Tong G, Wenger D, Lipton SA, Seyfried TN, Platt FM, Snyder EY. Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease. Nature Med 2007;13:439-47.

Summarized by: Pamela Orellana and Denis Chang, Fall 2007

LAY SUMMARY

Neural stem cells (NSCs) have a range of therapeutic actions in neurodegenerative diseases. This study investigated the benefit of neural stem cells in a mouse model of Sandhoff disease, which results from a deletion of the b-chain in b-hexosaminidase (Hex), causing deficiencies in the isoenzymes HexA and HexB leading to accumulation of gangliosides within lysosomes throughout the central nervous system. Children with this disease have severe mental retardation and motor dysfunction and death usually occurs in infancy.

Four different NSCs were transplanted separately into the forebrain and cerebellum of newborn Hex deficient mice: 1) mouse NCSs (mNCSs) from a stable, clonal population of engraftable lacZ-expressing mNSCs from clone C17.2 mice, 2) mNSCs isolated as neurospheres from the telencephalon of E10.5 Rosa 26 mice. 3) human NSCs (hNCSs) isolated from the telencephalic ventricular zone of late first-trimester human fetal cadavers, 4) hNSCs derived in vitro from human embryonic stem cells. In all four trials, results showed that symptoms onset was delayed and lifespan was prolonged, an improvement of ~70% compared to untreated age-matched mice. Motor functions was assessed by rotarod performance and the righting reflex test and showed that transplanted mice performed better than untreated mice.

Mechanism of improvement was concluded to be due to NSCs cross correcting β-hexosaminidase deficiency, which reduces monosialoganglioside (GM2) and gangliotriaosylceramide (GA2) storage. This was demonstrated using immunochemistry to assess the distribution and content in the cerebral cortex of Hex deficient mice transplanted at birth with mouse NSCs. Both GM2 and GA2 were significantly reduced in the mouse model. The impact of GM2/GA2 storage was also tested using human NSCs and it showed similar results.

Furthermore, this study suggests that the effectiveness of the cross corrective enzyme supplied by the NSCs can be improved by reducing the amount of substrate to be metabolized. In substrate reduction therapy (SRT), imino sugars, N-butyldeoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ), are used to inhibit GM2/GA2 biosynthesis and storage. When transplantation of mouse NSCs into neonatal cerebrum was combined with SRT administration, the Hex deficient mice survival was increased by 67-71%.

Transplanted NSCs migrated throughout the Hex deficient mouse, providing the cross correcting enzyme, and reducing ganglioside storage and inflammation. In combination with other therapies, it might serve as a better prognosis in neurodegenerative disorders.  

SCIENTIFIC SUMMARY

SD is caused by a deletion of the b-chain in b-hexosaminidase (Hex), resulting in deficiencies in the isoenzymes HexA (ab) and HexB (bb) and accumulation of gangliosides within lysosomes throughout the central nervous system (CNS). Four different NSCs, two mouse NSCs (mNSCs) and two human NSCs (hNSCs), were transplanted individually into the forebrain and cerebellum of newborn Hexb-/- SD mice. One population of mNSCs was a stable, clonal population of engraftable lacZ-expressing mNSCs from clone C17.2 mice while the other was mNSCs isolated as neurospheres from the telencephalon of E10.5 Rosa 26 mice. mNSCs from clone C17.2 was selected due to their over expression of the stemness-like gene myc, which preserved multipotency, self-renewal, and the undifferentiated state in vitro. “Primary” hNSCs were isolated from the telencephalic ventricular zone of late first-trimester human fetal cadavers and maintained in serum-free medium containing bFGF, heparin, and LIF. “Secondary” hNSCs were derived in vitro from hESCs and maintained under feeder-free culture conditions. Following transplantation, a marked improvement was observed in all cases. Improved motor function was determined by rotarod performance and righting reflex test while the onset of symptoms was delayed along with prolonged survival (Figure 1).

To investigate the potential mechanism underlying these results, the distribution of engraftment and the degree to which the replacement of mutant with wild-type neural cells were studied. Though engraftment in the hindbrain was less robust, the evident distribution of donor-derived cells in the forebrain pointed towards neuronal replacement as the mode of action. This, however, was discredited as being the primary cause following the similarities that existed in the degree of improvement of motor function and lifespan between the administration mNSCs prenatally and neonatally, the former resulting in more cortical neuron production in comparison with the latter.

NSCs were shown to improve motor function and delayed symptom onset by reconstituting Hex enzyme activity, as they are known to express normal levels of Hex constitutively, and by reducing microglial activation and macrophage infiltration, both being hallmarks of SD pathogenesis. The cross-corrective enzyme decreased levels of glycosphingolipid gangliotriaosylceramide (GA2) and monosialoganglioside (GM2) storage in SD mice, while the NSCs suppressed levels of CD11b, F4/80, and CD68, which are markers of microglia/macrophage activation.

Similarly, trials using both primary and secondary hNSCs resulted in marked improvement in both aspects of motor function and prolonged survival. Results also suggest similar causes for improvement rather than neuronal replacement following transplant. In all the experiments involving both the mNSCs and hNSCs, immunosuppressants were not required, despite the transplants being an allograft and xenograft, respectively.

Although the findings were only therapeutic, as the mice eventually became subjected to the disease, this study shows great potential, especially in cooperation with other treatments. It was shown that when the NSC transplant was synergized with substrate reduction therapy (SRT), in which imino sugars, specifically N-butyldeoxynojirimycin or N-butyldeoxygalactonojirimycin, are introduced orally, it resulted in greater improvements in motor function and significant delaying of symptom onset as compared to each mechanism utilized separately. Such strategies, along with others, may be used collaboratively to provide better prognoses in cases involving SD or other neuronal disorders.

Factors, such as the media in which the NSCs are harvested and cultured, and the environments under which the NSCs are subject to and grown must be considered in evaluating the validity of the results obtained in this study. As stem cells are sensitive to microenvironmental exposure, it is important to bear in mind the role of the cultures and the sources from which they are derived prior to transplant. Further studies involving the anti-inflammatory and immunosuppressive behavior of the NSCs would also be of interest as immunosuppresants were not required despite the transplants being an allograft and xenograft.

Figure 1
a.) Hexb-/- SD mice transplanted with fibroblasts, while failed to migrate from the lateral ventricles, exhibited a gradual decrease in rotarod performance throughout a 15-week period, a righting reflex test time of 9 seconds and survival for 114-130 days.  b.) Hexb-/- SD mice transplanted with NSCs exhibited an increase in motor function (improved rotarod performance and righting reflex test results) and prolonged survival (survival time of ~170 days).

Back