OT/Stem Cell Therapy for Autism

[Guest guest]

Journal of Translational Medicine

Open Access

Stem Cell Therapy for Autism

Abstract

Autism spectrum disorders (ASD) are a group of neurodevelopmental conditions

whose incidence is reaching epidemic proportions, afflicting approximately 1

in 166 children. Autistic disorder or autism is the most common form of ASD.

Although several neurophysiological alterations have been associated with

autism, immune abnormalities and neural hypoperfusion appear to be broadly

consistent. These appear to be causative since correlation of altered

inflammatory responses, and

hypoperfusion with symptology is reported. Mesenchymal stem cells (MSC) are

in late phases of clinical development for treatment of graft versus host

disease and Crohn's Disease, two conditions of immune dysregulation. Cord

blood CD34+ cells are known to be potent angiogenic stimulators, having

demonstrated positive effects in not only peripheral ischemia, but also in

models of cerebral ischemia. Additionally, anecdotal clinical cases have

reported responses in autistic children receiving cord blood CD34+ cells. We

propose the combined use of MSC and cord blood CD34+cells may be useful in

the treatment of autism.

Background

Autism spectrum disorders (ASD) are reaching epidemic proportions, believed

to affect approximately 1 in 166 children. Autism, Asperger's syndrome,

Rett's disorder, and childhood disintegrae disorder are all encompassed by

the term ASD. Autism is the most prevalent ASD, characterized by

abnormalities in social interaction, impaired verbal and nonverbal

communication, and repetitive, obsessive behavior. Autism may vary in

severity from mild to disabling and is believed to arise from genetic and

environmental factors. While symptomology of autism may be noted by

caregivers around 12–18 months [1], definitive diagnosis generally occurs

around 24–36 months, however in some cases diagnosis may be made into

adulthood. Determination of autism is performed using the DSM-IV-TR, or

other questionnaires and tests. Children with autism appear withdrawn,

self-occupied, and distant. Inflexibility in terms of learning from

experiences and modifying patterns to integrate into new environments is

characteristic of autism. Depending on degree of severity, some children

with autism may develop into independent adults with full time employment

and self sufficiency; however this is seldom the case. Current treatments

for autism can divided into behavioral, nutritional and medical approaches,

although no clear golden standard approach exists. Behavioral interventions

usually include activities designed to encourage social interaction,

communication, awareness of self, and increase attention. Nutritional

interventions aim to restrict allergy-associated dietary components, as well

as to supplement minerals or vitamins that may be lacking. Medical

interventions usually treat specific activities associated with autism. For

example, serotonin reuptake inhibitors (SSRI's) such as fluoxetine,

fluvoxamine, sertraline, and clomipramine, are used for treatment of anxiety

and depression. Some studies have shown that SSRI's also have the added

benefit of increasing social interaction and inhibiting repetitive behavior.

Typical antipsychotic drugs such as thioridazine, fluphenazine,

chlorpromazine, and haloperidol have been showed to decrease behavioral

abnormalities in autism. Atypical antipsychotics such as risperidone,

olanzapine and ziprasidone have also demonstrated beneficial effect at

ameliorating behavioral problems. Autism associated seizures are mainly

treated by administration of anticonvulsants such as carbamazepine,

lamotrigine, topiramate, and valproic acid. Attention

deficient/hyperacti vity is treated by agents such as methylphenidate

(Ritalin®). Currently, numerous clinical trials are being conducted with

interventions ranging from hyperbaric oxygen, to administration of zinc, to

drugs exhibiting anti-inflammatory properties. Unfortunately, no clear

understanding of autism's pathogenic mechanisms exists, and as a result

numerous strategies are being attempted with varying degrees of success. In

this paper we examine two pathologies associated with autism – hypoperfusion

to the brain and immune dysregulation – and propose a novel treatment: the

administration of CD34+ umbilical cord cells and Mesenchymal cells.

Hypoperfusion of brain in autism

Children with autism have been consistently shown to have impaired, or

subnormal CNS circulation, as well as resulting hypoxia. Defects include

basal hypoperfusion, and decreased perfusion in response to stimuli that

under normal circumstances up regulates perfusion. In numerous studies the

areas affected by hypoperfusion seem to correlate with regions of the brain

that are responsible for functionalities that are abnormal in autism. For

example, specific temporal lobe areas associated with face recognition,

social interaction, and language comprehension, have been demonstrated to be

hypoperfused in autistic but not control children. The question of cause

versus effect is important. If temporal lobe ischemia is not causative but

only a symptom of an underlying process, then targeting this pathology may

be non-productive from the therapeutic perspective. However this appears not

to be the case. It is evident that the degree of hypoperfusion and resulting

hypoxia correlates with the severity of autism symptoms. For example,

statistically significant inverse correlation has been demonstrated between

extent of hypoxia and IQ. Supporting a causative effect of hypoperfusion to

autism development, Bachavelier et al reviewed numerous experimental reports

of primate and other animal studies in which damage causing hypoperfusion of

temporal areas was associated with onset of autism-like disorders. It is

also known that after removal or damage of the amygdala, hippocampus, or

other temporal structures induces either permanent or transient

autistic-like characteristics such as unexpressive faces, little eye

contact, and motor stereotypies occurs. Clinically, temporal lobe damage by

viral and other means has been implicated in development of autism both in

adults, and children. Evidence suggests that hypoperfusion and resulting

hypoxia is intimately associated with autism, however the next important

question is whether reversion of this hypoxia can positively influence

autism. In autism the associated hypoxia is not predominantly apoptotic or

necrotic to temporal neurons but associated with altered function.

Hypoperfusion may contribute to defects not only by induction of hypoxia but

also allowing for abnormal metabolite or neurotransmitter accumulation.

This is one of the reasons why glutamate toxicity has been implicated in

autism and a clinical trial at reversing this using the inhibitor of

glutamate toxicity, Riluzole, is currently in progress. Conceptually the

augmentation of perfusion through stimulation of angiogenesis should allow

for metabolite clearance and restoration of functionality. Although not

well defined, cell death may also be occurring in various CNS components of

autistic children. If this were the case, it is possible that neural

regeneration can be stimulated through entry of neuronal progenitor cells

into cell cycle and subsequent differentiation. Ample evidence of neural

regeneration exists in areas ranging from stroke, to subarachinoidal

hemorrhage, to neural damage as a result of congenital errors of metabolism.

Theoretically, it is conceivable that reversing hypoxia may lead to

activation of self-repair mechanisms. Such neural proliferation is seen

after reperfusion in numerous animal models of cerebral ischemia. The

concept of increasing oxygen to the autistic brain through various means

such as hyperbaric medicine is currently being tested in 2 independent

clinical trials in the US. However, to our knowledge, the use of cell

therapy to stimulate angiogenesis has not been widely used for the treatment

of autism.

Immune deregulation in autism

The fundamental interplay between the nervous system and the immune system

cannot be understated. Philosophically, the characteristics of self/non self

recognition, specificity, and memory are only shared by the immune system

and the nervous system. Physically, every immune organ is innervated and

bi-directional communication between neural and immune system cells has been

established in numerous physiological systems. In autism, several

immunological abnormalities have been detected both in the peripheral and

the central nervous systems. Astroglial cells, or astrocytes, surround

various portions of the cerebral endothelium and play a critical role in

regulating perfusion, and blood brain barrier function. Astrocytes are

capable of ediating several immunological/ inflammatory effects. Expression

of various toll like receptors (TLR) on astrocytes endows the ability to

recognize not only bacterial and viral signals but also endogenous " danger "

signals such as heat shock proteins, fibrinogen degradation products, and

free DNA. Physiologically, astrocytes play an important protective role

against infection, generating inflammatory cytokines such as TNF-alpha,

IL-1beta, and IL-6. Through secretion of various chemokines such as CXCL10,

CCL2 and BAFF, astrocytes play an important role in shaping adaptive immune

responses in the CNS. Astrocytes have antigen presenting capabilities and

have been demonstrated to activate T and B cell responses against exogenous

and endogenous antigens. Although astrocytes play a critical role against

CNS infection, these cells also have potential to cause damage to the host

when functioning in an aberrant manner. For example, various neurological

diseases are associated with astrocyte overproduction of inflammatory

agents, which causes neural malfunction or death. In amyotrophic lateral

sclerosis (ALS), astrocyte secretion of a soluble neurotoxic substance has

been demonstrated to be involved in disease progression. Astrocyte

hyperactivation has been demonstrated in this disease by imaging, as well as

autopsy studies. In multiple sclerosis, astrocytes play a key role in

maintaining autoreactive responses and pathological plaque formation. In

stroke, activated astrocytes contribute to opening of the blood brain

barrier, as well as secrete various neurotoxic substances that contribute to

post infarct neural damage. Vargas et al compared brain autopsy samples

from 11 autistic children with 7 age-matched controls. They demonstrated an

active neuroinflammatory process in the cerebral cortex, white matter, and

notably in cerebellum of autistic patients both by immunohistochemistr y and

morphology. Importantly, astrocyte production of inflammatory cytokines was

observed, including production of cytokines known to affect various neuronal

functions such as TNF-alpha and MCP-1. CSF samples from living autism

patients but not controls also displayed upregulated inflammatory cytokines

as demonstrated by ELISA. The potent effects of inflammatory cytokines on

neurological function cannot be underestimated. For example, patients

receiving systemic IFN-gamma therapy for cancer, even though theoretically

the protein should not cross the blood brain barrier, report numerous

cognitive and neurological abnormalities. In fact, IFNgamma, one of the

products of activated astrocytes, has been detected at elevated levels in

the plasma of children with autism. Mechanistically, inflammatory mediators

mediate alteration of neurological function through a wide variety of

different pathways, either directly altering neuron activity or indirectly.

For example, the common neurotoxin used in models of Parkinson's Disease,

MPTP is believed to mediate its activity through activation of IFN-gamma

production, leading to direct killing of dopaminergic neurons in the

substantia nigra. This is evidenced by reduced MPTP neuronal toxicity in

IFN-gamma knockout mice or by addition of blocking antibodies to IFN-gamma.

In terms of indirect effects of IFN-gamma, it is known that this cytokine

activates the enzyme 2, 3-indolaminedeoxyge nase, leading to generation of

small molecule neurotoxins such as the kynurenine metabolites 3OH-kynurenine

and quinolinic acid which have been implicated in dementias associated with

chronic inflammatory states. T cell and B cell abnormalities have been

reported systemically in autistic children. These have included systemic T

cell lymphopenia, weak proliferative responses to mitogens, and deranged

cytokine production. At face value, lymphopenia would suggest general immune

deficiency and as a result little inflammation, however, recent studies have

demonstrated that almost all autoimmune diseases are associated with a state

of generalized lymphopenia (reviewed by Marleau and Sarvetnick).

Autoimmune-like pathophysiology appears to be prevalent in autism and

several lines of reasoning suggest it may be causative. Firstly, numerous

types of autoantibodies have been detected in children with autism but not

in healthy or mentally challenged controls. These include antibodies to

myelin basic protein, brain extracts, Purkinje cells and gliadin extracted

peptides, neutrophic factors, and neuron-axon filament and glial fibrillary

acidic protein. Secondly, family members of autistic children have a higher

predisposition towards autoimmunity compared to control populations. Hinting

at genetic mechanisms are observations that specific HLA haplotypes seem to

associate with autism. Another genetic characteristic associated with autism

is a null allele for the complement component C4b. Both HLA haplotypes as

well as complement component gene polymorphisms have been strongly

associated with autoimmunity. It is known that autoimmune animals have

altered cognitive ability and several neurological abnormalities. Thirdly,

autism has been associated with a peculiar autoimmune-like syndrome that is

still relatively undefined. Mucosal lesions in the form of chronic

ileocolonic lymphoid nodular hyperplasia characterized by lymphocyte

infiltration, complement deposition, and cytokine production have been

described uniquely to children with autism but not healthy controls or

cerebral palsy patients. This inflammatory condition is associated not only

with lesions on the intestinal wall, but also in the upper GI tract.

Although several characteristics of this condition are shared with Crohn's

Disease, one unique aspect is eosinophilic infiltrate, which seems to be

associated with dietary habits of the patient. Systemic manifestation of the

immune deregulation/ chronic inflammatory condition are observed through

elevated levels of inflammatory cytokines such as IFN-gamma, IL-12, and

TNF-alpha. Indication that a relevant inflammatory response is ongoing is

provided by observation that the macrophage product neopterin is observed

elevated in children with autism. Inhibited production of anti-inflammatory

cytokines such as IL-10 and TGF-beta has also been observed in children with

autism, thus suggesting not only augmentation of inflammatory processes but

also deficiency of natural feedback inhibitor mechanisms. The systemic

effects of a chronic inflammatory process in the periphery may result in

production of soluble factors such as quinilonic acid, which have neurotoxin

activity. Ability of cellular immune deregulation to affect neural function

can occur independent of cell trafficking, as was demonstrated in animal

studies in which T cell depletion was associated with cognitive loss of

function that was reversible through T cell repletion [79]. Localized

inflammation and pathological astrocyte activation has been directly

demonstrated to be associated with pathogenesis in autism. Clinical trials

of inflammatory drugs have demonstrated varying degrees of success. For

example, in an open labeled study of the anti-inflammatory PPARgamma agonist

pioglitozone in 25 children, 75% reported responses on the aberrant behavior

checklist. Other interventions aimed at reducing inflammation such as

intravenous immunoglobulin administration reported inconsistent results,

however a minor subset did respond significantly. Clinical trials are

currently using drugs off-label for treatment of autism through inhibiting

inflammation such as minocycline, n-acetylcysteine, or ascorbic acid and

zinc. Despite the desire to correct immune deregulation/ chronic inflammation

in autism, to date, no approach has been successful.

Treatment of hypoperfusion defect by umbilical cord blood CD34+ stem cell

administration

Therapeutic angiogenesis, the induction of new blood vessels from

preexisting arteries for overcoming ischemia, has been experimentally

demonstrated in peripheral artery disease, myocardial ischemia, and stroke.

Angiogenesis is induced through the formation of collateral vessels and has

been observed in hypoperfused tissues. This process is believed to be

coordinated by the oxygen sensing transcription factor hypoxia inducible

factor-1 (HIF-1). During conditions of low oxygen tension, various

components of the transcription factor dimerize and coordinately translocate

into the nucleus causing up regulation of numerous cytokines and proteins

associated with angiogenesis such as SDF-1, VEGF, FGF, and matrix

metalloproteases. The potency of tissue ischemia stimulating angiogenesis is

seen in patients after myocardial infarction in which bone marrow angiogenic

stem cells mobilize into systemic circulating in response to ischemia

induced chemotactic factors. The angiogenic response has also been

demonstrated to occur after cerebral ischemia in the form of stroke and is

believed to be fundamental in neurological recovery. For example, in models

of middle cerebral artery occlusion, endogenous angiogenesis occurs which is

also involved in triggering migration of neural stem cells into damaged area

that participate in neuroregeneration. The association between neural

angiogenesis and neurogenesis after brain damage is not only

temporally-linked but also connected by common mediators, for example, SDF-1

secreted in response to hypoxia also induces migration of neural

progenitors. Angiogenic factors such as VEGF and angiopoietin have been

implicated in post ischemia neurogenesis. While recovery after cerebral

ischemia occurs to some extent without intervention, this recovery is can be

limited. Methods to enhance angiogenesis and as a result neurogenesis are

numerous and have utilized approaches that up regulate endogenous production

of reparative factors, as well as administration of exogenous agents. For

example, administration of exogenous cytokines such as FGF-2,

erythropoietin, and G-CSF, has been performed clinically to accelerate

healing with varying degrees of success. A promising method of increasing

angiogenesis in situations of ischemia is administration of cells with

potential to produce angiogenic factors and the capacity to differentiate

into endothelial cells themselves. Accordingly, the use of CD34+ stem cells

has been previously proposed as an alternative to growth factor

administration. Therapeutic administration of bone marrow derived CD34+

cells has produced promising results in the treatment of end-stage

myocardial ischemia, as well as a type of advanced peripheral artery disease

called critical limb ischemia [99]. Additionally, autologous peripheral

blood CD34+ cells have also been used clinically with induction of

therapeutic angiogenesis. Of angiogenesis stimulating cell sources, cord

blood seems to possess CD34+ cells with highest activity in terms of

proliferation, cytokine production, as well as endothelial differentiation.

Cord blood has been used successfully for stimulation of angiogenesis in

various models of ischemia. In one report, the CD34+, CD11b+ fraction, which

is approximately less than half of the CD34+ fraction of cord blood, was

demonstrated to possess the ability to differentiate into endothelial cells.

In another report, VEGF-R3+, CD34+ cells demonstrated the ability to

differentiate into endothelial cells and were able to be expanded 40-fold

expansion. The concentration of this potential endothelial progenitor

fraction in cord blood CD34+ cells is approximately tenfold higher as

compared to bone marrow CD34+ cells (1.9% +/- 0.8% compared to 0.2% +/-

0.1%) [103]. Administration of cord blood CD34+ cells into immune

compromised mice that underwent middle cerebral artery ligation reduced

neurological deficits and induce neuroregeneration, in part through

secretion of angiogenic factors. Several studies have confirmed that

systemic administration of cord blood cells is sufficient to induce

neuroregeneration. Given the potency of cord blood CD34+ cells to induce

angiogenesis in areas of cerebral hypoperfusion, we propose that this cell

type may be particularly useful for the treatment of autism, in which

ischemia is milder than stroke induced ischemia, and as a result the level

of angiogenesis needed is theoretically lower. However at face value,

several considerations have to be dealt with. Firstly, cord blood contains a

relatively low number of CD34+ cells for clinical use. Secondly, very few

patients have access to autologous cord blood; therefore allogeneic cord

blood CD34+ cells are needed if this therapy is to be made available for

widespread use. There is a belief that allogeneic cord blood cells can not

be used without immune suppression to avoid host versus graft destruction of

the cells. Numerous laboratories are currently attempting to expand cord

blood CD34+ cells, achieving varying degrees of success. Expansion methods

typically involve administration of cytokines, and or feeder cell layers.

The authors have developed a CD34+ expansion protocol that yields up to

60-fold expansion with limited cell differentiation.

This expansion method involves numerous growth factors and conditioned

medium, however is performed under serum free conditions (manuscript in

preparation) . Currently over 100 patients have been treated by one of the

authors with expanded CD34+ cells under local ethical approval with varying

degrees of success. Since other groups are also generating CD34+ expansion

technologies, we do not anticipate number of CD34+ cells to be a problem.

Safety concerns regarding allogeneic CD34+ cells are divided into fears of

graft versus host reactions, as well as host versus graft. The authors of

the current paper have recently published a detailed rationale for why

administration of cord blood cells is feasible in absence of immune

suppression [111]. Essentially, GVHD occurs in the context of lymphopenia

caused by bone marrow ablation. Administration of cord blood has been

reported in over 500 patients without a single one suffering GVHD if no

immune suppression was used. Although host versus graft may conceptually

cause immune mediated clearing of cord blood cells, efficacy of cord blood

Cells in absence of immune suppression has also been reported. Accordingly,

we believe that systemic administration of expanded cord blood derived CD34+

cells may be a potent tool for generation of neoangiogenesis in the autistic

brain.

Immune modulation by Mesenchymal stem cells

The treatment of immune deregulation in autism is expected to not only cause

amelioration of intestinal and systemic symptomology, but also to profoundly

influence neurological function. Reports exist of temporary neurological

improvement by decreasing intestinal inflammation through either antibiotic

administration or dietary changes. Although, as previously discussed, some

anti-inflammatory treatments have yielded beneficial effects, no clinical

agent has been developed that can profoundly suppress inflammation at the

level of the fundamental immune abnormality. We believe Mesenchymal stem

cell administration may be used for this purpose. This cell type, in

allogeneic form, is currently in Phase III clinical studies for Crohn's

disease and Phase II results have demonstrated profound improvement.

Mesenchymal stem cells are classically defined as " formative pluripotential

blast cells found inter alia in bone marrow, blood, dermis and periosteum

that are capable of differentiating into any of the specific types of

Mesenchymal or connective tissues. These cells are routinely generated by

culture of bone marrow in various culture media and collection of the

adherent cell population. This expansion technique is sometimes used in

combination with selection procedures for markers described above to

generate a pure population of stem cells. An important characteristic of

Mesenchymal stem cells is their ability to constitutively secrete immune

inhibitory factors such as IL-10 and TGF-b while maintaining ability to

present antigens to T cells. This is believed to further allow inhibition of

immunity in an antigen specific manner, as well as to allow the use of such

cells in an allogeneic fashion without fear of immune-mediated rejection.

Antigenspecific immune suppression is believed to allow these cells to shut

off autoimmune processes. Further understanding of the immune inhibitory

effects of Mesenchymal stem cells comes from the fact that during T cell

activation, two general signals are required for the T cell in order to

mount a productive immune response, the first signal is recognition of

antigen, and the second is recognition of costimulatory or coinhibitory

signals. Mesenchymal cells present antigens to T cells but provide a

coinhibitory signal instead of a co-stimulatory signal, thus specifically

inhibiting T cells that recognize them, and other cells expressing similar

antigens. Supporting this concept, it was demonstrated in a murine model

that Mesenchymal stem cell transplantation leads to permanent donor-specific

immunotolerance in allogeneic hosts and results in long-term allogeneic skin

graft acceptance. Other studies have shown that Mesenchymal stem cells are

inherently immunosuppressive through production of PGE-2, interleukin- 10 and

expression of the tryptophan catabolizing enzyme indoleamine 2,

3,-dioxygenase as well as Galectin-1. These stem cells also have the

ability to non-specifically modulate the immune response through the

suppression of dendritic cell maturation and antigen presenting abilities.

Immune suppressive activity is not dependent on prolonged culture of

Mesenchymal stem cells since functional induction of allogeneic T cell

apoptosis was also demonstrated using freshly isolated, irradiated,

Mesenchymal stem cells. Others have also demonstrated that Mesenchymal stem

cells have the ability to preferentially induce expansion of antigen

specific T regulatory cells with the CD4+ CD25+ phenotype. Supporting the

potential clinical utility of such cells, it was previously demonstrated

that administration of Mesenchymal stem cells inhibits antigen specific T

cell responses in the murine model of multiple sclerosis, experimental

autoimmune encephalomyelitis, leading to prevention and/or regression of

pathology. Safety of infusing Mesenchymal stem cells was illustrated in

studies administering 1–2.2 × 106 cells/kg in order to enhance engraftment

of autologous bone marrow cell. No adverse events were associated with

infusion, although level of engraftment remained to be analyzed in

randomized trials. The ability of Mesenchymal stem cells on one hand to

suppress pathological immune responses but on the other hand to stimulate

hematopoiesis leads to the possibility that these cells may also be useful

for treatment of the defect in T cell numbers associated with autism.

Practical clinical entry

We propose a Phase I/II open labeled study investigating combination of cord

blood expanded CD34+ cells together with Mesenchymal stem cells for the

treatment of autism. Such a trial would utilize several classical

instruments of autism assessment such as the Aberrant Behavior Checklist and

the Vineland Adaptive Behavior Scale (VABS) for assessment of symptomatic

effect. Objective measurements of temporal lobe hypoperfusion, intestinal

lymphoid hypertrophy, immunological markers and markers of hypoxia will be

included. In order to initiate such an investigation, specific

inclusion/exclusion criteria will be developed taking into account a

population most likely to benefit from such an intervention. Criteria of

particular interest would include defined hypoxia areas, as well as frank

clinical manifestations of inflammatory intestinal disease. Markers of

inflammatory processes may be used as part of the inclusion criteria, for

example, elevation of C-reactive protein, or serum levels of TNF-alpha,

IL-1, or IL-6 in order to specifically identify patients in whom the

anti-inflammatory aspects of stem cell therapy would benefit. More stringent

criteria would include restricting the study to only patients in which T

cell abnormalities are present such as ex vivo hypersecretion of interferon

gamma upon anti-CD3/CD28 stimulation, as well as deficient production of

immune inhibitory cytokines such as IL-10 and TGF-beta. One of the authors

has utilized both CD34+ and Mesenchymal stem cells clinically for treatment

of various diseases. In some case reports, the combination of CD34+ and

Mesenchymal stem cells was noted to induce synergistic effects in

neurological diseases, although the number of patients are far too low to

draw any conclusions. We propose to conduct this study based on the

previous experiences of our group in this field, as well as numerous other

groups that have generated anecdotal evidence of stem cell therapy for

autism but have not published in conventional journals. We believe that

through development of a potent clinical study with appropriate endpoints,

much will be learned about the pathophysiology of autism regardless of trial

outcome.

Click below to review the entire document including references; from the

Journal of Translational Medicine published June of 2007 titled " Stem Cell

Therapy for Autism "

http://www.translat ional-medicine. com/content/ pdf/1479- 5876-5-30. pdf