autologous bone marrow mononuclear cell therapy for autism...

Hindawi Publishing CorporationStem Cells InternationalVolume 2013, Article ID 623875, 13 pageshttp://dx.doi.org/10.1155/2013/623875

Clinical Study

Autologous Bone Marrow Mononuclear Cell Therapy forAutism: An Open Label Proof of Concept Study

Alok Sharma,1 Nandini Gokulchandran,1 Hemangi Sane,2 Anjana Nagrajan,3

Amruta Paranjape,3 Pooja Kulkarni,2 Akshata Shetty,3 Priti Mishra,3 Mrudula Kali,3

Hema Biju,3 and Prerna Badhe1

1 Department of Medical Services and Clinical Research, NeuroGen Brain and Spine Institute,Surana Sethia Hospital and Research Centre, Sion Trombay Road, Chembur, Mumbai 400071, India

2Department of Research & Development, NeuroGen Brain and Spine Institute, Surana Sethia Hospital and Research Centre,Sion Trombay Road, Chembur, Mumbai 400071, India

3Department of NeuroRehabilitation, NeuroGen Brain and Spine Institute, Surana Sethia Hospital and Research Centre,Sion-Trombay Road, Chembur, Mumbai 400071, India

Correspondence should be addressed to Pooja Kulkarni; [email protected]

Received 4 May 2013; Revised 24 June 2013; Accepted 7 July 2013

Academic Editor: Chen Lin

Copyright © 2013 Alok Sharma et al. his is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Cellular therapy is an emerging therapeutic modality with a great potential for the treatment of autism. Recent indings show thatthe major underlying pathogenetic mechanisms of autism are hypoperfusion and immune alterations in the brain. So conceptually,cellular therapy which facilitates counteractive processes of improving perfusion by angiogenesis and balancing inlammation byimmune regulation would exhibit beneicial clinical efects in patients with autism. his is an open label proof of concept studyof autologous bone marrow mononuclear cells (BMMNCs) intrathecal transplantation in 32 patients with autism followed bymultidisciplinary therapies. All patients were followed up for 26months (mean 12.7). Outcomemeasures used were ISAA, CGI, andFIM/Wee-FIM scales. Positron Emission Tomography-Computed Tomography (PET-CT) scan recorded objective changes. Out of32 patients, a total of 29 (91%) patients improved on total ISAA scores and 20 patients (62%) showed decreased severity on CGI-I.he diference between pre- and postscores was statistically signiicant (� < 0.001) on Wilcoxon matched-pairs signed rank test.On CGI-II 96% of patients showed global improvement. he eicacy was measured on CGI-III eicacy index. Few adverse eventsincluding seizures in three patients were controlled with medications.he encouraging results of this leading clinical study providefuture directions for application of cellular therapy in autism.

1. Introduction

Autism spectrum disorders (ASD) are a group of het-erogeneous neurodevelopmental disorders characterized bydeicits in verbal and nonverbal communication, socialinteraction, and presence of stereotypical repetitive behav-ior. he genetic, environmental, and immunological factorshave been attributed as underlying causes, though its exactetiology is unknown. he incidence of autism has increasedto a great extent, which may be due to increased awarenessleading to an early and accurate diagnosis or due to perinatal

complications, genetic factors, environmental factors, andlifestyle changes. Presently, the worldwide incidence is 12per 1000 children [1]. Despite its increasing rate, currentlyautism remains untreatable. he available options of behav-ioral therapy and pharmacological and supportive nutritionaltreatments are only palliative. Medical therapy is directedtowards the neuropsychiatric disorders associatedwithASDs.Commonly prescribed medicines are selective serotoninreuptake inhibitors, antipsychotics, mood stabilizers, andpsychostimulants. Methylphenidate may be used to treatattention deicit or hyperactivity. Anticonvulsants are used

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for seizures with autism [2]. However, the use of medicationsis limited by their side efects. here is a desperate needof a medical intervention to tackle the basic pathogeneticmechanisms. Several genes have been found to be associatedwith ASD. his provides the basis for treatment with genetherapy in future. Currently, for safe human gene therapyto be applied to this population, several areas need furtherresearch [3].

he major neurophysiological alterations are immuneabnormalities and neural hypoperfusion, and its correlationwith symptomatology has been reported [4]. Cellular therapyexerts potent angiogenetic and immunoregulatory efectsalong with other paracrine efects [5, 6]. Recently, cell trans-plantation has been shown to be safe and eicacious in severalneurological disorders [7–10]. A variety of cellular therapieswith diferent cell types and routes of administration is beingexplored. he major types are embryonic, umbilical cord,induced pluripotent, and adult stem cells. he use of adultstem cells is devoid of any ethical issues and can be obtainedfrom bone marrow, adipose tissue, skin, dental pulp, andother sources. Bone marrow stem cells have been extensivelystudied and can be easily procured [8, 11]. he intrathecalroute is an easy, safe, and direct approach to provide the cellsto the brain without causing any neural tissue damage [12, 13].

Conceptually, cellular therapy should give beneicial clin-ical efects in patients with autism. he concept is basedon its potential to counterbalance the core pathogeneticmechanisms of autism [4]. he aim of this study is to assessthe safety, eicacy, and clinical efects of autologous bonemarrow mononuclear Cells (BMMNCs) transplantation inpatients with autism.

2. Materials and Methods

2.1. Study Design. his is an open label proof of conceptstudy on the use of autologous BMMNCs transplantation in32 patients with autism. he intervention included cellulartherapy with intrathecal transplantation of autologous bonemarrow derived mononuclear cells followed by occupationaltherapy, speech therapy, and psychological intervention. heprimary objective was to document any adverse events andestablish the safety of the intervention within a period of 26months (December 2010 to February 2013). he secondaryobjective of the study was to evaluate the efects of the inter-vention on symptoms, disease severity, extent of disability,and functional impairment caused by autism.

2.2. Participants Eligibility Criteria and Recruitment. Patientselection was based on World Medical Association HelsinkiDeclaration for Ethical Principles for medical researchinvolving human subjects [14]. A written informed consentwas obtained from the parents of all patients. All the patientsincluded in the study had conirmed diagnosis of autismaccording to the DSM-IV TR diagnostic criteria for autis-tic disorder. he exclusion criteria were presence of acuteinfections such as HIV/HBV/HCV, malignancies, bleedingtendencies, renal failure, severe liver dysfunction and other

acute medical conditions such as respiratory infection andpyrexia.

2.3. Intervention

2.3.1. Preintervention Assessment. he Institutional Commit-tee for Stem Cell Research and herapy (IC-SCRT) grantedthe ethical approval for the treatment protocol. An informedconsent was taken from the parents of all the patients. Priorto intervention, all the patients underwent a thorough clinicalexamination with serological, biochemical, and hematolog-ical tests. Magnetic Resonance Imaging (MRI) of the brainand Electroencephalography (EEG) were also conducted inall patients. In view of the clinical improvements observed,a preintervention PET-CT (Positron Emission Tomography-Computed Tomography) scan was introduced at a later stageof the study.

2.3.2. Procurement of Autologous BMMNCs. Patients wereadministered Granulocyte Colony Stimulating Factor(GCSF) 48 hours and 24 hours before the harvest andtransplantation of BMMNC [15]. On the day of thetransplantation, bone marrow was aspirated under generalanesthesia in the operation theatre with aseptic precautions.Approximately, 100mL of bone marrow (varying between80mL and 100mL, based on the age and body weight) wasaspirated from the region of anterior superior iliac spineusing the bone marrow aspiration needle and collected inthe heparinized tubes.

2.3.3. Isolation of BMMNCs. he aspirate was then trans-ferred to the laboratory where the mononuclear cells wereseparated by the density gradient method. CD34+ countingwas done by Fluorescence activated cell sorting (FACS) [16].heMNCs were checked for viability (average viability countwas found to be 97%).

2.3.4. Mode of Cell Transplantation. he separated autol-ogous BMMNCs were immediately injected on the sameday, intrathecally using an 18G Tuohy needle and epiduralcatheter at the level between fourth and ith lumbar verte-brae. he average numbers of cells injected were 8.19 × 107.Simultaneously 20mg/kg body weight methyl prednisolonein 500mL Ringer Lactate was given intravenously to enhancesurvival of the injected cells. Patient was monitored for anyadverse events.

2.3.5. Post-BMMNCs Transplantation herapy. All thepatients underwent extensive therapy under the guidanceof experts. his includes occupational therapy interventionsbased on sensory integrative approach, activities of dailyliving (ADL) training, psychological intervention basedon behavior modiication techniques, speech therapy, andspeciic dietary recommendations. he therapy protocol wasplanned out speciically for individual patients, as per thedetailed assessment done before the therapy.

he therapy sessions conducted during the stay in thehospital were recorded and compiled in a CD-ROM which

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was given to all the patients at discharge. A therapy planto be followed at home was designed for all the patients.Patients were advised to continue therapy at home under thesupervision of a professional. Follow-up assessment was doneat regular intervals over a period of 26 months.

2.4. Outcome Measures. To assess the safety of the interven-tion, outcome measures were used to monitor any major orminor adverse events through the entire duration of followup.Patients were counseled regarding the probable adverseevents during informed consent. Recording of adverse eventsduring the hospital stay was done by a health professionalwhereas ater discharge it was recorded, as reported by theparents or primary care givers.

2.4.1. Monitoring Procedure Related Adverse Events. Acuteprocedural adverse events, associated with cell aspiration andinjection via lumbar puncture, were stringently monitoredover 5–7 days ater intervention. Body temperature, bloodpressure, respiratory rate, and heart rate were recorded at reg-ular intervals. Patients were examined thoroughly for signsof spinal headache, motor or sensory loss, incontinent boweland bladder, damage to brain or spinal cord, respiratorydistress, cardiac failure, and allergic reaction. Aspiration andinjection sites were examined every day for pain, bleeding,and signs of infection. Signs and symptoms of any anesthesiacomplications, back pain associated with lumbar puncture,headache, nausea, and vomiting were checked regularly. Allthe minor acute procedural adverse events were treatedmedically prior to the discharge of the patients from thehospital. Patients were examined thoroughly at each followupfor neurological deicits exhibiting clinically as motor orsensory loss that was not present before intervention. Adetailed history was also taken to rule out any transientneurological symptoms.

2.4.2. Monitoring Cellular Transplantation Related AdverseEvents. During the stay in the hospital, signs and symptomsof any allergic reaction were monitored at regular intervals.Long term major and minor adverse events were monitoredto establish the safety of stem cell transplantation. A detailedhistory was also taken to rule out presence of any seizures.If any seizure was reported, EEG was done for evaluationand managed by a neurologist. Any clinical deteriorationof the core symptoms of autism was recorded by IndianScale for Assessment of Autism (ISAA), Clinical GlobalImpression (CGI) scale, Functional Independence Measure(FIM), and Wee-FIM scales (see Supplementary Materialavailable online at doi: http://dx.doi.org/10.1155/2013/623875(Appendices I, II, III, and IV)).

2.4.3. Monitoring the Efects Ater Intervention. Outcomemeasures used for the efects of interventionwere CGI, ISAA,FIM, andWee-FIM scales. CGI scale was used for measuringthe change in the severity of the disease, overall improvement,and the eicacy of the treatment. he eicacy componentof CGI takes into consideration the improvements and side

efects ater the cellular transplantation. CGI-I scale for sever-ity of illness was scored before cellular therapy and at follow-up visit. CGI-II scale for global improvement and CGI-IIIscale for eicacy index were each measured at the time offollow-up assessment. he scores recorded by experiencedclinicians during the latest visit were used for the analysis.he CGI-I scale is an ordinal scale and relects a clinicalcomparison of patients’ severity of symptoms with the typicalclinical presentation. Although CGI scale is grossly perceivedas a subjective scale the psychometric properties of the CGIscale have been calculated earlier for various psychiatricdisorders. he scale shows good reliability, validity, andsensitivity for these disorders [17–19]. he CGI scale forautism has been previously used successfully as an outcomemeasure in various trials [20–22].

he efect of cell transplantation on the extent of dis-ability was measured using the ISAA. Most of the availablediagnostic tools and outcome measures have been designedfor the western population. In a disorder like autism wherecommunication and social behavior is impaired, an assess-ment tool that takes into account the social and culturalcontext is crucial. Although derived from CARS, ISAA hasbeen designed for Indian population. It is divided into sixdomains. here are forty questions which are comprehensiveand speciic to the diiculties experienced by children withautism. It grades the symptoms in ascending order of inten-sity of symptoms on an ordinal scale of 1 to 5. he content,construct and concurrent validity, internal consistency andtest-retest reliability, and sensitivity and speciicity of ISAAwere studied by the members of the expert committee forthe development of assessment tool for autism. ISAA wasthus found to be a valid tool, with good reliability and highsensitivity and speciicity [23]. ISAA scores were markedduring the assessment before the stem cell transplantationand at every follow-up assessment ater transplantation,however the scores marked at the latest assessment wereconsidered for comparison and analysis.

To measure the functional independence in activitiesof daily living, Wee-FIM for children below the age of 8years and FIM for above 8 years were used. FIM has beenshown to be a valid and reliable tool [24]. In addition tothese outcome measures various symptoms were monitoredthrough structured interviews and assessment by clinicians.

In view of clinical improvements, PET-CT scanning wasintroduced to observe functional neuroimaging changes inbrain. Measurements were taken before and six months aterthe transplantation. PET studies were performed using theSiemens Biograph mCT with 64-slice high speed scanner,3D PET True V wide detector (Siemens-CTI, Knoxville,Tenn., USA), which has an intrinsic resolution of 0.6mm fullwidth at half maximum (FWHM) and the images of 45–50contiguous transverse planes with a ield of view of 21.6 cmaxial PET FOV with True V.

Standard conditions were maintained during all of the

[18F1] FDG PET scans. Time duration between injectionof the dye and scanning was constant at 30 minutes forall the patients and at all instances. he scan room wasdimly lit and there was minimal auditory stimulation during


Table 1: Demographic data and scores of CGI, ISAA, FIM, and Wee-FIM scales before cell transplantation.

Category Minimum Maximum Average/median Standard deviation

Demographic data

Follow-up duration (months) 5 26 12.7 7.1

Age at intervention (years) 3 33 10.49 5.59

Diagnosed since (years) 0 18 7.17 4.20

Baseline scale scores

CGI-I scale scores 3 6 4.5 0.97

ISAA scale scores 148 160 115.5 24.26

FIM scale scores 48 118 77 18.32

Wee-FIM scale scores 18 110 76 24.06

injection and scanning period. PET scan was performed withpatients lying in supine position with eyes closed to reduceany activity related confounding efect. Imaging data wereprocessed using proprietary Scenium Sotware before inalimage reconstruction.

2.5. Statistical Analysis

2.5.1. Description of Sample. he demographic data for allthe patients was recorded and analyzed. Mean age in yearsat the time of intervention, mean age in years at the time ofdiagnosis, and mean time duration in months at which thepatients were followed up were calculated. Median CGI andFIM scores were calculated. Median score was calculated forthe total ISAA score and the individual domains.

2.5.2. Statistical Tests. he pre- and postintervention scoresof CGI-I, total ISAA scores, scores of ISAA domains, Wee-FIM, and FIM were compared using Wilcoxon signed ranktest for matched pairs with the predetermined level ofsigniicance at 0.05. Percentage analysis was conducted forthe CGI-II and CGI-III scales and for individual symptomsas described in ISAA scale. Statistical analysis was carried outusing SPSS 17.0 sotware.

3. Results

3.1. Description of the Sample. here were total 32 patientswith 24 (75%) males and 8 (25%) females. he age of thepopulation ranged from 3 to 33 years with a mean age of 10.5(5.6) years. hey were diagnosed on an average at 7.1 (5.2)years before the intervention. he follow-up period rangedbetween 5 and 26 months with a mean followup of 12.7 (7.1)months. he baseline ISAA scores ranged from 148 to 160with a median of 115.5 (18.33) (due to the even number ofpatients), CGI-I scores ranged from 3 to 6 with a median of4.5 (1), FIM scores ranged from 48 to 118 with a median of 77(18), andWee-FIM scores ranged from 18 to 118 with amedianof 76 (24) (Table 1).

3.2. Statistical Analysis. here was a statistically signiicantdiference between the pre- and post-CGI-I scores (� =0.001) and total ISAA scores (� < 0.001) (Table 2).here wasno statistically signiicant diference between the FIM scores

Table 2: Change in the scores of CGI and ISAA before and aterintervention.

Scale

Medianscore beforecellulartherapy

Median scoreater the

cellular therapy

Teststatistics

Statisticalsigniicance

CGI-I 4.5 3 � = −3.509 � < 0.001∗

ISAA scale 115.5 97 � = −4.670 � < 0.001∗∗Statistically signiicant (level of signiicance at � < 0.05).

(� = −1.841, � = 0.066) and Wee FIM scores (� = −1.000,� = 0.317). All the ISAA domains showed a statisticallysigniicant (� < 0.05) reduction ater intervention (Table 3).

3.3. Percentage Analysis. Overall the ISAA score reducedin 29 out of 32 (90.6%) patients. On CGI-II scale 96.9%showed global improvement. Out of these 43.7% patientsshowed much improvement and 34.4% patients showedvery much improvement. here were 18.8% with minimalimprovement and 3.1% patients with minimal worsening(Figure 1). According to the CGI-III scale 93.8% patients hadno side efects and only 6.2%patients hadminimal side efectsthat did not interfere with function. 21.8% patients showedmarked improvement with no side efects, 40.6% patientsshowed moderate improvement with no side efects, 28.1%patients showed minimal improvement with no side efects,3.1% patients showed marked improvement with side efectsthat did not interfere with patients functioning, and 3.1%patients showedmoderate improvement with side efects thatdid not interfere with patients functioning (Figure 2).

Here we describe the number of patients showingimprovement in a particular symptom as indicated by thepercentage in the parenthesis.

In the domain of social relationships and reciprocity, 29out of 32 (90.6%) patients showed improvement. Patientsshowed improved eye contact (70%), social smile (56%), andreaching out to others (42%). hey were able to take turns insocial interaction (55%), respond to social or environmentalcues (46%), andmaintain peer relationships (55%).herewasa decrease in three symptoms: inability to relate to people(30%), tendency to remain aloof (59%), and engagement insolitary and repetitive play activities (41%).


Table 3: Change in the ISAA scores of individual domains measured before and ater intervention.

ISAA scale domainMedian score before

cellular transplantationMedian score ater

cellular transplantation

Test statistics of Wilcoxonsigned rank test for matched

pairs

Statisticalsigniicance

Social relationship and reciprocity 35.5 13 −4.118 � < 0.001∗

Emotional responsiveness 23 20 −3.153 � = 0.002∗

Speech, language, and communication 13 11 −3.989 � < 0.001∗

Behavior patterns 29 10 −3.126 � = 0.002∗

Sensory aspects 21 17 −2.409 � = 0.016∗

Cognitive component 11 8 −3.508 � < 0.001∗∗Statistically signiicant (level of signiicance at � < 0.05).

0

2

4

6

8

10

12

14

16

Notassessed

Very muchimproved

Muchimproved

Minimallyimproved

Minimallyworse

Muchworse

Very muchworse

3.1%

18.8%

43.7%

34.4%

Nu

mb

er o

f p

atie

nts

Frequency distribution of participants on CGI-II scale

Nochange

CGI-II scores

Figure 1: Frequency distribution of participants on CGI-II scale.his igure shows the percentage of people showing varying degreesof improvement ater cellular transplantation.

Improved emotional responsiveness was observed in 18out of 32 (56%) patients. Inappropriate emotional responses(42%), exaggerated emotions (48%), engaging in self-stimulating emotions (55%), and getting excited or agitatedfor no apparent reason (56%) decreased. Lack of fear ofdanger (37%) did not reduce signiicantly; yet, few patientswere reported to have shown some decrease.

Under the speech, language, and communication domainthere was an improvement observed in 25 patients out of32 (78%). A signiicant reduction was seen in echolalicspeech (54%), engaging in stereotyped repetitive use oflanguage (53%), production of infantile squeals or unusualnoises (52%), inability to initiate or sustain conversation withothers (45%), and inability to grasp the pragmatics of theconversation (43%) and speech regression (50%). It is note-worthy that few patients also showed clinical improvementin symptoms of diiculty in using nonverbal language orgestures (32%), using of jargon or meaningless words (28%),using of pronoun reversal (27%).

Behavior patterns of 21 out of 32 patients (66%) improved.Hyperactivity or restlessness (71%) and engaging in stereo-type and repetitive motor mannerisms (65%) decreasedsigniicantly. Attachment to inanimate objects (50%), throw-ing temper tantrum (42%), aggressive behavior (48%), self-injurious behavior (53%), and insisting on sameness (43%)also reduced.

Marked improvement

Moderate improvement

Minimal improvement

Unchanged or worse

0

2

4

6

8

10

12

14

No sideefects Side efects

do notsigniicantly

interferewith patientsfunctioning

Side efectssigniicantly

interferewith patientsfunctioning

Side efectsoutweigh

therapeuticefects

3.1%

3.1%

3.1%

21.8%

40.6%

28.1%

Figure 2: Frequency distribution of the participants on the CGI-IIIscale. his igure demonstrates the percentage of people on varyingdegrees of eicacy of the intervention. his takes into considerationthe side efects and the beneits observed.

Sensory aspects improved in 14 out of 32 patients (44%).Unusual sensitivity to sensory stimuli (50%), staring intospace for long periods of time (25%), diiculty in trackingobjects (46%), unusual vision (62%), insensitivity to pain(26%), and responses to objects or people unusually bysmiling or touching or tasting (39%) reduced signiicantly.

Cognitively they showed improved consistency in atten-tion and concentration and response time. 71% patientsshowed better attention and concentration, 45% patientsshowed reduction in the delay in responding.

Functional neuroimaging in the form of PET-CT scanin eight patients noted the following changes. Ater cellulartherapy changes in the glucose metabolism in the form ofFDG uptake were observed in the frontal and parietal lobesof six patients, occipital and temporal lobes, of ive patientsand cerebellum of four patients. Further analysis of otherregions showed changes in medial temporal lobe of fourpatients; amygdala, hippocampus, and parahippocampus ofthree patients; and cingulate, paracingulate area, and basalganglia of ive patients.


Table 4: Adverse events monitored over the entire period of follow-up of 26 months.

Adverseevents

Present during the period of follow-up Absent during the period of follow-up

Procedure related Cellular transplantation related Procedure related Cellular transplantation related

Minor

(i) Spinal headache (3.6%)(ii) Nausea (10.7%)(iii) Vomiting (17.9%)(iv) Pain at the site ofinjection (7.1%)(v) Pain at the site ofaspiration (7.1%)

None

(i) Bleeding at the site ofinjection(ii) Bleeding at the site ofaspiration

None

Major None

(i) Seizures∗ (9%)(ii) Transient increase inhyperactivity (18.7%)(iii) Persistent increase inhyperactivity till six months(3.1%)

(i) Neurological deicits(ii) Nerve root damage(iii) Parasthesia in lower limb(iv) Loss of sensation in lowerlimb(v) Loss of motor function inthe lower limbs(vi) Hematoma at the site ofinjection(vii) Hematoma at the site ofaspiration(viii) Local infection at the siteof injection or aspiration(ix) Meningismus ormeningitis(x) Systemic or brain infection(xi) Bowel or bladderincontinence(xii) Respiratory distress(xiii) Cardiac failure

Allergic reaction

∗Seizures were considered to be an adverse event when seizures observed were new onset postintervention with no previous history or there was increasedfrequency or severity of seizures as compared to preintervention.

3.4. Adverse Events Monitoring

3.4.1. Procedure Related Adverse Events. During the proce-dure there were no complications in the operation theatre.None of the patients had signs and symptoms of local orsystemic infection, meningismus, any neurological deicit,parasthesias in the lower limb, any nerve root damage, orhematoma at the site of aspiration and allergic reaction.During the hospital stay, few patients showed procedurerelated minor adverse events like spinal headache (3.6%),nausea (10.7%), vomiting (17.9%), backache and pain at thesite of injection (7.1%), and aspiration (7.1%). hese adverseevents were controlled with medication and resolved withinone week. here were no procedure related major adverseevents (Table 4).

3.4.2. Cellular Transplantation Related Adverse Events.Adverse events related to cellular transplantation weretransient minimal increase in hyperactivity (6 out of 32),persistent increase in hyperactivity (1 out of 32), andseizures (3 out of 32). Six patients had transient increasein hyperactivity at three-month followup. One patientshowed persistent increase in hyperactivity at six monthsthat did not interfere with the global clinical improvement.One patient with marked symptomatic improvement andone patient with moderate symptomatic improvement

developed seizures ater therapy, which were controlled withantiepileptic medications. One out of four patients who hada previous history of seizures showed increased episodesof seizures for a few weeks which were controlled withincreased dosages of his antiepileptic medications (Table 5).Despite this the patient showed moderate improvementswith no symptomatic deterioration. Patients who had normalEEG before cellular transplantation did not have seizuresater cellular transplantation.

4. Discussion

An important aim of regenerativemedicine is to ind a deini-tive treatment for incurable disorders. In this efort, variousconditions such as Parkinson’s disease, spinal cord injuries,muscular dystrophy, myocardial infarction, and stroke arestudied and have shown beneicial therapeutic efects [7–9].Recently, cell-based therapy for autism has been explored byresearchers to a great extent [4–6].

Autism is one of the most complicated neurodevelop-mental disorders with a very high prevalence rate [1]. Its exactetiology and pathophysiology remains poorly understood.he numerous biochemical abnormalities detected in autismare oxidative stress, endoplasmic reticulum stress, mitochon-drial dysfunction, decreased methylation, underproductionof glutathione, intestinal dysbiosis, and toxic metal burden


Table 5: Details of three patients who had seizures as an adverse event ater cellular therapy.

Patient I Patient II Patient III

Type of seizures (before and aterintervention)

Pre-GTCPost-GTC

Pre-No seizurePost-GTC

Pre-No seizuresPost-GTC

Number of seizure episodes aterintervention

One Multiple Two

Duration in months between thecellular therapy and irst seizureepisode

Six Four hree

Duration in months over whichseizure episodes recurred

No recurrence Ten hree

Medication used for seizurecontrol

Midazolam

Sodium Valproate dose wasdoubled. Clobazam wasdiscontinued and Lamotriginewas added

Sodium Valproate

Seizure related complications None None None

Efect of seizures on clinicalimprovement

here was no deterioration inthe baseline and the markedclinical improvement wasmaintained



[25]. he environmental factors like organophosphates andheavy metals are also attributed to the origin of the disease[26]. Genetics involve multiple mutations in various genesresulting in its varied phenotype. hese mutations mayresult in structural or molecular or functional defects insynaptogenesis [27].

A range of indings have suggested autism as a disorderof growth of the neural systems and connections, likely tobe responsible for the underdevelopment of functions suchas communication, behavior, and socialization [28]. U. Frithand C. Frith (2010) proposed a social brain hypothesis toexplain theory of mind deicits in ASD [29]. he social brainconcept tries to localize the complex social perception tosuperior temporal sulcus (STS), amygdala, orbital frontalcortex (OFC), and fusiform gyrus (FFG) [30]. he key rolesimplicated are STS region in analysis of perception, FFG inface detection and recognition, OFC in social reinforcementand reward processes, and the amygdala in analysis andregulation of emotions [31]. hese areas form neural inter-connections to establish a pathway from perception to action[32]. Neuroimaging studies have indicated dysfunction in thesocial brain areas and aberrant neuronal circuitry in autism[33, 34].

Recently, brain hypoperfusion and immune dysfunctionhave been recognized as twomajor pathogeneticmechanismsassociated with autism. Hypoperfusion results in hypoxia,abnormalmetabolite or neurotransmitter accumulation lead-ing to neural tissue damage. he degree of hypoperfusionis proportional to the severity of the symptoms of autism.he extent of hypoxia was shown to be inversely correlatedto Intelligent Quotient (IQ) [35]. Immune dysfunction isan imbalance in pro-inlammatory and anti-inlammatoryfactors. he raised macrophage product neopterin, TNF-alpha, MCP-1, and IFN-gamma indicate an augmentedinlammatory response. In addition, deicient levels of anti-inlammatory cytokines such as IL-10 and TGF-beta suggest

lack of natural inhibitory feedback processes. Autoimmunemechanism is also thought to be causative due to detectionof autoantibodies to myelin basic protein, Purkinje cells andgliadin extracted peptides, neurotrophic factors, and neuron-axon ilament and glial ibrillary acidic protein [36, 37]. Tcell and B cell abnormalities have been demonstrated withsystemic T cell lymphopenia, decreased cell proliferation, andabnormal production of cytokines [38]. During the periodof neurodevelopment, the deregulated immune activity mayresult in the neurological dysfunctions in autism [39, 40].Ichim et al. (2007), in their review, have proposed theadministration of stem cells as a novel treatment to addressthe core pathologies of autism [4].

Cellular therapy has the therapeutic potential to repairthe damaged neural tissue at molecular, structural, andfunctional levels. he stem cells possess unique properties ofself-renewal, transdiferentiation [41], and paracrine efects[15]. he paracrine action changes the local micromilieu bysecretion of trophic factors like ciliary neurotrophic factor(CNTF), vascular endothelial growth factor (VEGF), andibroblast growth factor (FGF). his stimulates the localrepair by enhancing proliferation, cell recruitment, and mat-uration of endogenous stem or progenitor cells [42]. heCD34+ stem cells have the capacity to produce angiogenicfactors anddiferentiate into endothelial cells themselves [43].herapeutic angiogenesis improves perfusion and clearanceof toxic metabolites and reduces hypoxia.

Another important efect is immunomodulation throughinhibition of T lymphocyte pro-inlammatory cytokine pro-duction (IL-1�, TNF-�, and INF-�) and upregulation of anti-inlammatory IL-10 and TGF-beta. his counterbalances theaberrant immune systems and reduces neural damage withrestoration of functions [44, 45].

4.1. Source Selection and Route of Administration. Severalsources of stem cells have been identiied such as fetal or


embryonic, bone marrow, umbilical cord, adipose tissue anddental pulp [46]. Bone marrow derived stem cells are easilyprocured by a standard procedure [47] and its potency andsafety has been well established without any ethical issues[8, 9]. he bone marrow comprises of a heterogeneouspopulation of stem cells, encompassing hematopoietic stemcells (HSCs), mesenchymal stem cells (MSCs), endothelialprogenitor cells (EPCs), and very small embryonic-like stemcells (VSELs) [48]. his ofers advantage of variety efects ofdiferent cell types.

he choice of intrathecal route was guided by eicientdelivery of cells to brain with a relatively less invasive and safeprocedure. he injected cells are transported by CSF to theafected areas in the brain [49, 50]. Various mechanisms havebeen believed to lead to altered permeability of blood brainbarrier allowing the transplanted cells to reach brain areaswith marked inlammation [51, 52]. he intrathecal routeenhances the possibility of maximal number of transplantedcells “homing” onto damaged sites.

4.2. Clinical Findings inhis Study. Conceptually the cellulartherapy mechanisms, as described above, address the corepathogenesis of autism. he novel indings on the molecular,cellular, neuroimmunological, and environmental factorscontributing to the pathogenesis of autism provide a rationalefor cellular therapy as a unique and potent tool. herefore, asa proof of concept, we studied 32 cases of autism, which weretreated with BMMNCs, intrathecally.

he study sample included a total of 32 participants ofwhich 24 were males and 8 were females, which is a ratio of3 : 1. he gender ratio is synonymous to indings in previousstudies [53]. he study included children as well as adultswith autism. A majority of the patients were undergoingrehabilitation therapy since the time of diagnosis. Severityof autism in the participants ranged from mild to severe, asmeasured on the ISAA.

he clinical results, stated above as evidence to theconcept, are discussed here. With a good number of par-ticipants showing clinically signiicant improvements on theISAA and its subcomponents, the patterns of symptomaticimprovement were analyzed. We gather here a theoreticalpattern of the improvements noted ater cellular therapy.Immediately ater the intervention, within one week, initi-ation or consistency of eye contact and minimal decreasein hyperactivity were observed. Restlessness, rocking, handlapping, and jumping, which are motor behavior seen inrelation to the sensory issues, were seen to reduce early aterintervention and continue to do so even later.his aids in bet-ter participation during sessions for behavior managementand speech therapy. Only a few patients showed an increasein the levels of hyperactivity which could be attributed to thechallenges posed by a new social and physical environmentat the hospital and the changes in their routine. Arousaland activity levels continued to normalize progressively overthe next three to six months. he immediate efect of thedecrease in hyperactivity is seen on the levels of attention andconcentration. Attention span increases and patients begin tosit at one place for activities, attend to commands, and follow

them. hese enhance the quality and duration of therapysessions attended and aids in learning new concepts. Withthe ability to follow commands emerging and the improvedattention span, facilitation of meaningful communicationbecomes more efective. hey later start communicatingtheir needs and using nonverbal gestures too. Besides thesensory issues, behavior has also been attributed to theirinability to communicate or express. It was observed that,as they start communicating and indicating their needs andwith the appropriate reinforcement strategies, their abnormalbehavior slowly fades away. hey also began to initiate socialinteraction and engage in play, forming peer relationships.

Speechwas found to have improved at later stages. Speechis a complex function requiring good listening skills, atten-tion, auditory processing, comprehension, and motor co-ordination. All of these are afected in many individuals withautism. We believe that it requires longer duration of consis-tent therapy for signiicant improvements. Developmentallyspeech develops as monosyllable, bisyllables, words, phrasesand lastly sentences. We have observed that in the patientstreated with autologous BMMNCs followed by supportivetherapies that individuals who were absolutely nonverbal (ormute) before therapy developed nonverbal communication.Individuals who had monosyllables progressed to bisyllablesand so on (Figure 3).

Hence, signiicant improvements were noted in all theinterrelated domains of the ISAA, namely, social relationshipsand reciprocity, emotional responsiveness, speech, language,and communication, behavior patterns, sensory aspects, andcognition. Sensory aspects continually resolved and formedthe basis for behavior, followed by social interaction andresponsiveness, cognition, and speech or communication.

In each of the domains, a few symptoms did not improvein a large percentage of patients. he ability to relate topeople requires recognition, understanding of relations (e.g.,father and son), emotional bonding, and reciprocity. Onlya few patients (29.63%) showed improvement in this owingto a shorter duration of followup. On the three symptoms,namely, lack of fear of danger under the domain of emotionalresponsiveness, insensitivity to pain within the domain ofsensory aspects, and staring into space for long periods oftime; 36.84%, 26.32%, and 25% of individuals improved,respectively.We hypothesize that these are interrelated symp-toms. For emotional responsiveness, one may require todevelop a clear concept of self and the need to protect oneself.his may lead to the identiication and perception of a threatwhich requires higher levels of understanding (cognition)and conditioning for building the association between causeand efect.

Fear of danger is dependent on the perception of painwhich may be afected due to sensory processing and inte-gration problems. hey may be underresponsive to tactile,vestibular, and proprioceptive inputs. Yet another possibilityis their increased attention towards details and intricacies dueto which they miss out on the gross information (e.g., theymay be so engrossed in looking at a car’s wheels or designso that they fail to notice it speeding towards them). hisintense observation of minute features or objects could be theunderlying cause of what seems to us as staring into space.


Eye contact

develops/improves

Hyperactivity

decreases

Repetitive motor

mannerisms decrease

Attention span improves

Cooperation and active

participation in therapy

sessions increase

Conveying needs

and expressing

self improve

Sensory issues

decrease

Improved learning

and concept

formation

Nonverbal

communication/

gestures improve

Verbal communication

improves

Social interaction

improves

Behavioral

issues

decrease

Speech and language

improve

Global life-skills development

Command

following

improves

Communication

improves

Figure 3: Schematic representation of clinical improvements ater cellular therapy.his igure shows proposed theoretical outline of observedchanges ater cellular therapy.

Under the speech, language, and communicationdomain, few individuals improved on the use of nonverballanguage (31.58%), decreased use of jargon or meaninglesswords (27.78%), and decreased pronoun reversals (27.27%).As stated before speech and communication require longerperiods of training for individuals with autism. For reducingthe use of jargon, the child must understand that what heor she is saying is meaningless and inappropriate. his maynot be handled with behavior strategies alone. A higher levelof cognition needs to be developed. Pronoun reversals arelinked to the deicit in self-identity and the concept of “I”,“Me,” or “You,” which is afected in them. With a longerduration of followup these areas may get addressed.

Within the domain of cognition, unusual memory andsavant ability were unchanged as they are intrinsic to theindividual with autism and have a complex underlyingmechanism of development.

On FIM and Wee-FIM scales there was no statisticallysigniicant change, as the FIM scores were maintained atercellular transplantation suggesting preserved independencelevel for ADL in all the patients ater cellular therapy.

4.3. heoretical Basis for the Clinical Improvements Seen aterthe Intervention. Kevin et al. presented a model of ASD thatimplicates an early failure to develop the specialized functionsof the social brain that is involved in social information


processing. hey state that due to this early disruption, “anindividual with autism must develop in a highly social worldwithout the specialized neural systems that would ordinarilyallow him or her to partake in the fabric of social life,which is woven from the thread of opportunity for socialreciprocity and the tools of social engagement [30].” Wehypothesize that cellular transplantation causes functionalrestoration of specialized neural systems by neuroprotection,neural circuit reconstruction, neural plasticity, neurogenesis,and immunomodulation. Individual therapies like occupa-tional therapy, psychological intervention, and speech ther-apy employ the principles of learning to facilitate neuralplasticity. In addition, they also provide the opportunity andtools for social engagement. Enhancement of the neural andfunctional restoration can be optimized by combining thesetherapies with cellular transplantation.

4.4. Adverse Events. here were some minor adverse events,such as headache (3.6%), nausea (10.7%), vomiting (17.9%),pain at the site of injection (7.1%), and pain at the siteof aspiration (7.1%) which were reported in few patientsimmediately ater cell transplant procedure. hese mild sideefects were controlled with medications and resolved withina week. It is important to consider poor communication skillsof children with autism while monitoring the symptomaticadverse events.

One (3.1%) patient was marked as minimally worse onCGI-II, due to persistent increase in hyperactivity at threemonths which did not subside by six months of followup.During this study, we observed that 6 patients (18.7%) showeda transient phase of minimal increase in hyperactivity in theirst three months that subsided by six months. In additionthis increase in the hyperactivity did not interfere with theoverall improvement. he transient increase in hyperactivitymay be a result of increased neuronal activity ater celltransplantation, changes in daily routine, and exposure tonew tasks during therapy sessions. his needs to be furtherstudied in larger sample and controlled studies.

In few patients (9%) there were seizure episodes atercellular transplantation which were controlled with medica-tions. his may be due to activation of epileptogenic focuswhich already existed in these patients. his is supportedby absence of seizures ater intervention in the patientswith normal EEG befor intervention. It was observed thatin patients with no history of seizures but EEG reportingepileptogenic focus, therewas increased likelihoodof seizuresater intervention. It is noteworthy that the seizures did notcause clinical deterioration or hamper beneicial efects of celltherapy.

No othermajor adverse events were noted over the periodof 26 months (mean = 12.7 months).

he overall improvements on ISAA was observed in 29patients out of 32 (90.6%), and 20 patients out of 32 (62.5%)improved on CGI-I, which was signiicant. he grading ofglobal improvements onCGI-II showed that 37.5% of patientshad verymuch improvement, 46.8%hadmuch improvement,and 18.8% had minimal improvement.

CGI-III eicacy index provides us with evidence that theintervention may be an efective tool for treatment of autism.21.9% of patients showed marked therapeutic efect with noside efects, 40.6% patients showed moderate therapeuticefect, and 28% showed minimal therapeutic efect; none ofthese patients had any major side efects.

4.5. PET-CT Scan Evidence. he complex constellation ofsymptoms of autism cannot be explained by pin pointing toa speciic structure or area of the brain. he hypothesis ofsocial brain and theory of mind tasks emphasizes that thesymptoms of autism are routed not only in the structuraldeviations but also in the deicits in neural connectivity[29]. Various attempts have been made to investigate theareas of dysfunction, using various radiological and nuclearimaging techniques [54, 55]. hese investigations, however,do not explain the putative neural connectivity deviationscausing the symptoms of autism. Functional neuroimagingis thought to give more lucid information about neuralconnectivity [56]. PET-CT scan and Functional MRI (FMRI)scan are most widely used functional neuroimaging tech-niques. PET-CT scan of brain is a noninvasive, relativelysafe, and feasible modality to record the functional activityof brain. It measures 18-FDG uptake which is related tothe glucose metabolism at the cellular level which correlateswith the functioning of the area of the brain. PET-CT scanremains a choice of investigation in children with autism dueto relative ease of conduction and measurement. PET-CTscan studies in children with autism have earlier identiiedreducedmetabolic activity in bilateral temporal lobes [57, 58].Another study showed signiicant hypoperfusion of superiortemporal gyrus and superior temporal sulcus in childrenwithautism compared to the control children [33]. hese indingsare consistent with the theory of social brain. We used PET-CT scan to observe the metabolic activity of the brain beforeand ater cellular therapy.he scanwas done in a standardizedmanner, maintaining similar conditions before scanning toameliorate confounding factors and therefore the changes inthe 18-FDG uptake may be attributed to the intervention.

A comparative PET-CT scan before and six monthsater cellular transplantation showed a balancing efect onthe metabolism. he areas of hypermetabolism implicatedin previous imaging studies [59] showed reduction inmetabolism ater cellular transplantation, and areas havinghypometabolism as suggested by Zilbovicius et al. 2000 [33]showed increased metabolism ater cellular transplantation.We hypothesize that immunomodulatory efects and neoan-giogenesis causes improved oxygenation and functioningof the damaged neurons. his improves their metabolismwhich leads to increased FDG uptake in the previouslyhypofunctional neurons. he paracrine efects and the anti-inlammatory efects also lead to inhibition of hyper func-tional neurons causing decrease in FDG uptake in thepreviously hyperfunctional neurons. he exact mechanismstill remains unknown. hese changes could be clinicallycorrelated with statistically signiicant reduction of scores ofthe domains of ISAA scale and reduction in the severity ofdisease as measured on CGI-I scale (Figure 4).


(a)

(b)

Figure 4: Findings in PET-CT scan before and ater cellular therapy. (a) PET-CT scan before intervention showing reduced FDG uptake inthe areas of frontal lobe, cerebellum, amygdala, hippocampus, parahippocampus, andmesial temporal lobe. (b) PET-CT scan sixmonths aterintervention comparison shows increased FDG uptake in the areas of frontal lobe, cerebellum, amygdala, hippocampus, parahippocampus,and mesial temporal lobe.

4.6. Limitations and Future Directions. he study is an openlabel proof of concept. A small sample size, the absenceof randomization, and the absence of control group werethe limitations. Large scale, multicentre, and randomizedcontrolled trials are recommended. A longer period of fol-lowup may be required to further establish the safety andeicacy. Few patients had increased episodes of seizures aterthe intervention, which were controlled with medications.We recommend preintervention EEG assessments to identifypatients with high risk for seizures, ater cellular therapy.herequirement and efectiveness of prophylactic antiepilepticmedications in such patients must be studied. PET-CT scanwas used as evidence in a small number of patients as itwas introduced at a later stage, in view of marked clinicalimprovements. Future studies should consider the use ofPET-CT scan as amonitoring tool and substantiate the efectsof cellular therapy in autism.

his study presents results of autologous BMMNCsintrathecal administration, still the other routes of adminis-tration; types of cells, combination of cells, dosage of cells,and frequency of the transplantation must be explored. Itis believed that the efect of stem cells is larger in youngerage group due to greater plasticity of the brain and increasedavailability of precursor cells in the bone marrow. It may behelpful to identify the most responsive age group for cellulartherapy.

In this study, one of our concerns was that the improve-ments observed may be due to various therapies other thancell transplantation. But, several studies have been reviewedfor evidence of efects of individual therapies includingoccupational therapy, sensory integration, behavior therapy,and speech therapy in children with autism. Case-Smith andArbesman in their review [60] show that these individualtherapies have some positive indings, but the evidence isweak due to limitations of small sample size, short fol-lowup, lack of evaluation instruments, inadequate measuresof treatment idelity, and inappropriate data analysis. So,according to them the interpretation of the indings of thestudies of the individual therapies needs to be with caution.herefore, the signiicant clinical improvements seen in ourstudy cannot be attributable to themultidisciplinary therapiesalone. Our study demonstrates that cellular therapy has syn-ergistic efect and enhances the efects of multidisciplinarytherapies. Hence, we postulate that the combination of celltransplantation with various therapies ofers an augmentedbeneicial response. Future studies may be planned with acontrol group for these individual therapies.

5. Conclusion

Despite accumulating evidence of the safety of cellular ther-apy, there is a dearth of published human clinical studies.


Autism has been in discussion since a long period andthe increasing incidence has established a need to ind adeinitive treatment modality. herefore, the transition ofcellular therapy from benchside to bedside is warranted.hough autologous BMMNCs may not be a cure for autism,they deinitely possess the potential tomanage overall diseaseseverity and improve the quality of life. his study is apreliminary demonstration of the safety and eicacy ofautologous BMMNCs in autism. he minimal invasiveness,simplicity of the procedure, and autologous nature of the cellsrender it as a promising therapeutic potential. his is the irstclinical study on the application of cellular therapy in patientswith autism which may provide future directions for largerrandomized controlled trials.

Conflict of Interests

he authors declare that they have no conlict of interests.

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