Abstract
BackgroundAmyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of MNs in the central nervous system. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Despite the selective MN death in ALS, there is growing evidence that malfunctional astrocytes play a crucial role in disease progression. Thus, transplantation of healthy astrocytes may compensate for the diseased astrocytes.MethodsWe developed a good manufacturing practice-grade protocol for generation of astrocytes from human embryonic stem cells (hESCs). The first stage of our protocol is derivation of astrocyte progenitor cells (APCs) from hESCs. These APCs can be expanded in large quantities and stored frozen as cell banks. Further differentiation of the APCs yields an enriched population of astrocytes with more than 90% GFAP expression (hES-AS). hES-AS were injected intrathecally into hSOD1G93A transgenic mice and rats to evaluate their therapeutic potential. The safety and biodistribution of hES-AS were evaluated in a 9-month study conducted in immunodeficient NSG mice under good laboratory practice conditions.ResultsIn vitro, hES-AS possess the activities of functional healthy astrocytes, including glutamate uptake, promotion of axon outgrowth and protection of MNs from oxidative stress. A secretome analysis shows that these hES-AS also secrete several inhibitors of metalloproteases as well as a variety of neuroprotective factors (e.g. TIMP-1, TIMP-2, OPN, MIF and Midkine). Intrathecal injections of the hES-AS into transgenic hSOD1G93A mice and rats significantly delayed disease onset and improved motor performance compared to sham-injected animals. A safety study in immunodeficient mice showed that intrathecal transplantation of hES-AS is safe. Transplanted hES-AS attached to the meninges along the neuroaxis and survived for the entire duration of the study without formation of tumors or teratomas. Cell-injected mice gained similar body weight to the sham-injected group and did not exhibit clinical signs that could be related to the treatment. No differences from the vehicle control were observed in hematological parameters or blood chemistry.ConclusionOur findings demonstrate the safety and potential therapeutic benefits of intrathecal injection of hES-AS for the treatment of ALS.
Highlights
Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of motor neurons (MNs) in the central nervous system
In animal ALS models, we show that intrathecal injection of hES-AS into the cerebrospinal fluid (CSF) of hSOD1G93A mice and rats had significant effects on delaying disease onset, maintaining motor performances and delayed death
The protocol was optimized to include good medical practice (GMP)-grade materials and factors to be compatible for clinical use
Summary
Amyotrophic lateral sclerosis (ALS) is a motor neuron (MN) disease characterized by the loss of MNs in the central nervous system. As MNs die, patients progressively lose their ability to control voluntary movements, become paralyzed and eventually die from respiratory/deglutition failure. Amyotrophic lateral sclerosis (ALS) is an adult-onset disease characterized by the loss of both upper and lower motor neurons (MNs). Symptoms include progressive paralysis of MN target muscles. The disease is incurable, and fatal within 3–5 years of first symptoms, due to respiratory failure when the diaphragm is affected [1]. About 10–15% of cases of ALS are familial, and the other cases are sporadic. Familial ALS includes mutations in Cu2+/Zn2+ superoxide dismutase-1 (SOD1) [2] and in RNA/DNA binding proteins FUS and TAR DNA binding protein-43 [3]. The most frequent genetic cause of ALS (40% of familial ALS) is an amplification of a hexanucleotide in a noncoding region of the C9orf gene [4]
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