The use of stem cells for the treatment of … SB, Braz J Med Biol Res...1579 Braz J Med Biol Res 40(12) 2007 Use of stem cells in autoimmune diseases Brazilian Journal of Medical

1579

Braz J Med Biol Res 40(12) 2007

Use of stem cells in autoimmune diseases

www.bjournal.com.br

Brazilian Journal of Medical and Biological Research (2007) 40: 1579-1597ISSN 0100-879X Rewiew

The use of stem cells for the treatmentof autoimmune diseases

1Laboratório de Hematologia, Faculdade de Farmácia,2Programa de Pós-graduação em Biologia Celular e Molecular,3Departamento de Genética, 4Programa de Pós-graduação em Medicina:Ciências Médicas, Faculdade de Medicina,Universidade Federal do Rio Grande do Sul, Porto Alegre, RS, Brasil5Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto,Universidade de São Paulo, Ribeirão Preto, SP, Brasil

S.B. Rosa1,2,J.C. Voltarelli5,

J.A.B. Chies3

and P. Pranke1,4

Abstract

Autoimmune diseases constitute a heterogeneous group of conditionscommonly treated with anti-inflammatory, immunosuppressant andimmunomodulating drugs, with satisfactory results in most cases.Nevertheless, some patients become resistant to conventional therapy.The use of high doses of drugs in such cases results in the need forbone marrow reconstitution, a situation which has stimulated researchinto the use of hematopoietic stem cells in autoimmune diseasetherapy. Stem cell transplantation in such diseases aims to destroy theself-reacting immune cells and produce a new functional immunesystem, as well as substitute cells for tissue damaged in the course ofthe disease. Significant results, such as the reestablishment of toler-ance and a decrease in the recurrence of autoimmune disease, havebeen reported following stem cell transplantation in patients withautoimmune disease in Brazil and throughout the world. These resultssuggest that stem cell transplantation has the potential to become animportant therapeutic approach to the treatment of various autoim-mune diseases including rheumatoid arthritis, juvenile idiopathicarthritis, systemic lupus erythematosus, multiple sclerosis, systemicsclerosis, Crohn’s disease, autoimmune blood cytopenias, and type Idiabetes mellitus.

CorrespondenceP. Pranke

Laboratório de Hematologia

Av. Ipiranga, 2752, 3º andar

90160-000 Porto Alegre, RS

Brasil

Fax: +55-51-3308-5437

E-mail: [email protected]

Research supported by FINEP

and CNPq.

Publication supported by FAPESP.

Received October 29, 2006

Accepted July 2, 2007

Key words• Autoimmune disease• Stem cells• Transplantation• Lupus erythematosus• Arthritis• Type I diabetes mellitus

Introduction

Autoimmune diseases (AD) are causedby immunologic imbalance and loss of toler-ance, which lead the immune system to at-tack self tissues within the organism. AD canbe mediated by immune complexes, circu-lating autoantibodies and autoreactive T lym-phocytes. Conventional AD therapy is effec-tive in most patients, but some patients areresistant to the anti-inflammatory and immu-

nosuppressive agents used or are only ca-pable of responding to high doses of suchmedicines, which are toxic. In such cases,bone marrow (BM) reconstitution is required.Thus, high doses of immunosuppressants,followed by hematopoietic stem cell trans-plantation (HSCT), have become an alterna-tive treatment for many diseases involvingthe immune system. These include multiplesclerosis (MS), systemic sclerosis (SS), rheu-matoid arthritis (RA), juvenile idiopathic ar-

1580


S.B. Rosa et al.

www.bjournal.com.br

thritis (JIA), and systemic lupus erythemato-sus (SLE) (1,2).

The application of HSCT to the treat-ment of AD has been studied since the 1970s.The success of this approach has been widelydemonstrated in animal models as well as inBM transplant patients who were shown toalso have concomitant AD. For example, anallogeneic BM transplantation (BMT), whichwas intended to cure aplastic anemia in 2patients with concomitant RA, resulted inthe complete remission of RA for at least 11years (2).

The use of stem cell therapy in diseasesinvolving the immune system is increas-ingly common. By 2004, the European andNorth American data banks had accumu-lated information on about 800 patientstreated with HSCT for a variety of diseases(3). Most of these procedures involved pe-ripheral blood autologous HSCT (AHSCT).After the administration of growth factors tothe patient, which mobilizes the stem cellsfrom BM to peripheral blood, the patient’sblood is collected and processed, and maturecells are excluded until a sufficient numberof HSCs are obtained for transplantation.The patient then undergoes radioactive and/or cytotoxic drug therapy, which eliminatethe cells in division, including immune sys-tem cells. The treated blood is returned to thepatient via transfusion, and, by a mechanismknown as homing, the HSCs migrate to theBM, where they begin a process of differen-tiation into mature functional cells. This treat-ment has been shown to result in the reestab-lishment of tolerance and a decrease in therecurrence of AD. Figure 1 shows a sche-matic representation of this procedure.

Stem cells

Stem cells are characterized by their ca-pacity for self-renewal, multilineage differ-entiation, proliferation, mobilization, andhoming (4). Stem cells can be divided intotwo main groups: embryonic stem cells

(ESCs) and adult stem cells (ASCs).ESCs, found in the embryo until approxi-

mately 3 days after fertilization, are totipo-tent, that is, they are capable of giving rise tomore than 200 cell types, including extra-embryonic tissues such as the placenta andumbilical cord. ESCs obtained from the in-ternal mass of cells that form the blastocyst,at 4 or 5 days following embryo fertilizationof the embryo, but before implantation in theuterus, are pluripotent cells (5). That is, theyare capable of giving rise to all cell types,with the exception of extra-embryonic tis-sues.

ASCs give rise to specific tissue progeni-tor cells and are, in most cases, multipotent,producing a more limited number of celltypes. The stem cells most commonly usedin clinical medicine are HSC, a type of mul-tipotent ASC, which give rise to all types ofblood and immune system cells (5). Themain sources of HSCs are BM, umbilicalcord blood (UCB) and, in smaller quantities,peripheral blood. Some ASCs appear to becapable of transdifferentiation. This processis characterized by the differentiation of aprogenitor cell into a cell line other than thatfrom which it originated. An example is thecapacity of BM stem cells to give rise toneurons (6). Later, pluripotent stem cells,such as mesenchymal stem cells (MSCs)and multipotent adult progenitor cells, werediscovered in several adult tissues.

In the next sections, we report on a num-ber of AD for which stem cell transplanta-tion is currently being considered a potentialtherapeutic approach. Additionally, we pro-vide examples of the different therapeuticprotocols used, and discuss the limitationsand advantages of such procedures.

Rheumatoid arthritis and juvenileidiopathic arthritis

Autologous stem cell transplantation(ASCT) has been proposed as a possibletreatment for severe AD such as RA, MS,

1581



www.bjournal.com.br

SS, and SLE. Almost 1000 patients withvarious autoimmune disorders have under-gone ASCT since 1996, among them a smallnumber of children, most of them sufferingfrom JIA (7).

RA is a systemic AD that, in the longterm, can lead to irreversible destruction ofthe joints, loss of mobility, as well as areduction in both the quality of life and lifespan. Cellular and humoral immune re-

sponses can contribute to the developmentof lesions. Rheumatoid factor, an autoanti-body specific to the Fc region of human IgG,is found in 80% of patients with RA (8).

The cells involved in inflammatory reac-tions are derived from HSCs, which sug-gests that this disease may originate fromproblems in stem cells. It is not clear whetherthe inflammatory cells are derived from ab-normal stem cells or whether they are nor-

Figure 1. Schematic model of the protocol for obtaining autologous hematopoietic stem cells from peripheral blood for transplantation showing thedifferent stages of transplantation, re-establishment of immunological tolerance and possible causes of relapse. Following granulocyte-colonystimulating factor (G-CSF) treatment which stimulates the mobilization of stem cells from bone marrow to peripheral blood (1), the peripheral blood stemcells are collected by means of leukapheresis and purified (2). Purification of peripheral blood via antithymocyte globulin (ATG) in vivo and/or in vitroselection of CD34+ cells aims to isolate stem cells from mature cells. After collection of peripheral blood, the patient is submitted to immunosuppressivetherapy (2). The graft containing the stem cells is reintroduced into the patient (4). If mature lymphoid cells remain in the graft after the purification stage,the patient may suffer a relapse (3B and 4B). The stem cells multiply via the self-renewal process (5) and are capable of differentiation (6), forming anew immunological system and therefore re-establishing immunological tolerance, consequently diminishing the symptoms of the disease or leading tolong-term remission (7A). However, if new auto-reactive cells develop during the differentiation stage or the purification stage fails to remove such cellsfrom the graft, the patient may suffer a relapse (7B).

Peripheral blood Peripheral blood

1) G-CSF

2) LeukapheresisGraft: ATG and/or CD34+ selectionPatient: immunosuppressive therapy

Pluripotent stem cell

Mature autoreactive lymphocyte

3A) Graft

4A) Re-introduction of the stemcells in the patient

4B) Re-introduction of the stemcells in the patient Relapse of the disease

5) Self-renewal

6) Differentiated cells

7A) Reestablishment ofimmunological tolerance

7B) Relapse ofthe disease6) Differentiated cells

5) Self-renewal

3B) Graft

1582


S.B. Rosa et al.

www.bjournal.com.br

mal progenitor cells that demonstrate patho-genicity within a microenvironment predomi-nantly occupied by inflammatory cytokines.Proof that the defect lies in stem cells is thefact that RA can be transmitted or abolishedby allogeneic stem cell transplantation (9).

One hypothesis concerning RA is that itinvolves the aging of stem cells. The HLA-DR4 haplotype appears to be linked to RA.Healthy HLA-DR4+ individuals present pre-mature aging of HSCs. Aged T cells expressdistinct immunoregulatory receptors thatpotentiate their activity, independently ofantigenic recognition. These cells presentpro-inflammatory properties and trigger auto-reactivity. In RA, the abnormal functioningof stem cells is probably not limited to HSCs,but may also involve, for example, MSCs.Thus, the premature aging of stem cells inRA may lead to tissue destruction resultingfrom the action of T cells and failure of thetolerance mechanism (10).

Study of the application of stem cells tothe treatment of RA started at the beginningof the 1980s in research using animal mod-els. Clinical studies mainly involve autolog-ous HSCs or, more rarely, syngeneic or allo-geneic BMT (11). The transplantation pro-tocol varies between studies. As in otherAD, in order to perform peripheral bloodAHSCT the cells are mobilized using granu-locyte-colony stimulating factor (G-CSF)with or without immunosuppression, priorto leukapheresis. The graft may or may notbe manipulated in vivo or ex vivo usingantithymocyte globulin (ATG) and/or byselection of CD34+ cells, the classic markerof HSCs. The purpose of these procedures isto eliminate mature autoreactive lympho-cytes, thus preventing them from being re-introduced into the organism. The patient issubmitted to an intense immunosuppressiveprocess followed by the reintroduction ofstem cells into the receptor. Several clinicalstudies are discussed throughout this reviewand are listed in Table 1. The patients se-lected in the following studies presented

severe RA which had proven to be resistantto conventional therapies.

Mobilization of HSCs with G-CSF isconsidered to be a suitable technique. How-ever, the administration of cyclophospha-mide (CY) prior to G-CSF appears to offersome advantages. Immunosuppression in-duced by CY may reduce the risk of intensi-fying the RA symptoms produced by G-CSF. CY seems to increase HSC levels,facilitating graft manipulation, and to re-duce the number of potentially pathogenic Tlymphocytes re-introduced into the patient.Moreover, it appears to stimulate renovationof immunocompetent cells, reestablishingthe immunological balance, without the needfor HSC re-infusion (12).

This conditioning regime should providegreat efficacy with reduced toxicity. Thus,the dose of CY to be used has been estab-lished as 200 mg/kg since it produces aneffective response in RA treatment and pre-sents acceptable levels of toxicity (13). In astudy involving 4 patients who received CYand ATG, 1 patient was submitted to totalbody irradiation (TBI), 2 patients, includingthe one that received TBI, presented diseasereactivation post-transplantation, though thesymptoms were mild and transitory. Theother 2 patients reached improvement levelACR70. According to the American Collegeof Rheumatology (ACR), ACR70 corre-sponds to an improvement level of 70% inthe count of joint pain and edema, togetherwith an improvement of 70% in at least 3 ofthe 5 parameters, including the level of reac-tive C protein, erythrocyte sedimentation,and functional incapacity. Similarly, ACR20and ACR50 correspond to improvements of20 and 50%, respectively (14).

Research into the use of high doses ofCY in the absence of stem cell re-infusion isbased on the HSC expression of high levelsof aldehyde dehydrogenase, an enzyme re-sponsible for the cellular resistance of CY(9), and 2 patients submitted to this proce-dure showed complete remission of the dis-

1583



www.bjournal.com.br

Table 1. Protocols of autologous transplantation and results of clinical studies based on the use of stem cells in the treatment of rheumatoid arthritis.

PBHSCwithout

re-infusionof cells

CY (4 g/m2) +G-CSF

Non-manipulatedcells

Group 1:4 patients:

CY 100 mg/kg

Group 2:4 patients:

CY 200 mg/kg

Results Conclusion References

· Improvement of symptoms,relapse in all patients.Symptoms did not affect pre-treatment intensity

· Increase in the number of TCD4+ cells in relation to theevolution of RA

Immuno-suppression

-

Manipulation

-

MobilizationCells used

4

Number ofpatients

8 PBHSC Filgrastim

· The use of highdoses of CY priorto G-CSF is safeand effective in mo-bilization

12

· Dose-dependent hematologi-cal toxicity

· Group 1: improvement insymptoms with relapse in 3-4months post-treatment

· Group 2: more intense andlong lasting improvement

· The use of 100 mgCY/kg is not recom-mended. 200 mg CY/kg produces effectiveresponses in RA andtoxicity levels are ac-ceptable

13

Selection ofCD34+ cells

CY (200 mg/kg)+ ATG

1 patient: TBI

4 PBHSC CY (2 g/m2)and G-CSF

· Improvement of all patients: 1failed to reach ACR20, 2reached ACR70

· 2 patients, including the one whoreceived TBI: reappearance ofthe disease, with mild and tran-sitory symptoms

· AHSCT: safe andeffective in 2 of 4patients

· TBI did not preventRA reactivation

14

Group A:18 patients:selection ofCD34+ cells

Group B:15 patients:

non-manipulatedstem cells

CY 200 mg/kg33 PBHSC Filgrastim · Reached ACR70: group A:27.7% and group B: 53.3%

· Average recurrence time: groupA: 147 days and group B: 201days

· Average RF (3 months): group A:219 IU/mL and group B: 30 IU/mL

· After recurrence (12 patients):67% spontaneous remission orwith DMARDS

· Selection of CD34+

cells for transplan-tation fails to showbenefits

· AHSCT appears toact as an immuno-modulator

16

28 patients:non-manipulated

cells

Other patients:selection ofCD34+ cells

and/orchemotherapy

CY

CY + ATG

CY + TBI + ATG

CY + Busulfan

Fludarabine +ATG

76 72 patientsPBHSC

3 patients:only stem cellmobilization

1 patient:stem cellscollectedfrom BM

G-CSF

G-CSF + CY(4 g/m2) ±etoposide

· Mobilization intensified the RAin 15% of the patients

· 3 patients: complete remission;33 patients: ACR70; 13 patients:ACR50; 12 patients: ACR20; 12patients: therapy ineffective

· Re-administration of DMARDS:disease controlled in half of thecases

· Better response in patients withnegative RF and absence of cor-relation with: the duration of thedisease, previous number ofDMARDS used, presence ofHLA-DR4 or removal of lympho-cytes from graft

· Therapy with highdoses of immuno-suppressor andHSCT is safe

· Most of the patientsresponded well, butwith relapse

· Stem cell trans-plantation is not acurative therapy,but it permits thecontrol of reactiva-tion of the diseasewith DMARDS

8(EBMT

andABMTR

from 1996to 2000)

The studies cited derive from a review of the literature and give a general view of the different outcomes after therapy. ABMTR = Autologous Blood andMarrow Transplant Registry; ACR = American College of Rheumatology level (in percent); AHSCT = autologous hematopoietic stem cell transplanta-tion; ATG = antithymocyte globulin; BM = bone marrow; CY = cyclophosphamide; DMARDS = disease-modifying antirheumatic drugs; EBMT =European Group for Blood and Marrow Transplantation; G-CSF = granulocyte-colony stimulating factor; HSCT = hematopoietic stem cell transplanta-tion; PBHSC = peripheral blood hematopoietic stem cells; RA = rheumatoid arthritis; RF = rheumatoid factor; TBI = total body irradiation.

1584


S.B. Rosa et al.

www.bjournal.com.br

ease (15). Moore and colleagues (16) stud-ied 33 patients who were randomly submit-ted to either the autologous transplantationof non-manipulated cells or the transplanta-tion of selected CD34+ cells. The effects ofusing non-manipulated stem cells were bet-ter than those obtained with selected CD34+

cells. In a syngeneic transplantation of HSCs,in which the patient with RA received HSCsfrom his twin brother, no recurrence of thedisease was seen for at least 24 months.There were some differences in the expres-sion of some T cell receptor subtypes be-tween the brothers. Following transplanta-tion, the T cell receptor repertory of the hostbecame practically the same as that of donor.Thus, the syngeneic lymphocytes from ahealthy donor are capable of modifying theimmunoregulation disorder, contributing tothe production of graft-versus-autoimmu-nity (17).

Most studies on HSCT involve isolatedcases or small sample groups. A wider study,based on the records of both the EuropeanGroup for Blood and Marrow Transplanta-tion (EBMT) and the Autologous Blood andMarrow Transplant Registry, involved thecollection of results from around the worldof cases of treatment of RA with stem cells,particularly severe and resistant cases. From1996 to 2000, 76 patients were registered.The mortality rate related to the treatmentwas low, with the death of 1 patient, who hadbeen submitted to graft purification and im-munosuppression. Most of the patients re-sponded well to the treatment, although witha high rate of relapse. AHSCT makes itpossible to control the return of the diseasewith the use of conventional antirheumaticdrugs. The good response to these post-transplantation drugs suggests that ASCT isassociated in some way with immunomodu-lation (8,13,14,16).

Thus, HSCT in RA therapy is well toler-ated by patients, with few cases of deathsrelated to transplantation. Currently, AHSCTis not considered to be a curative therapy for

RA. However, it may represent a possible“back to the beginning of the disease mech-anism”, facilitating the control of symptomintensification through the use of mainte-nance therapy (9). This post-transplantationmaintenance has been the focus of the EBMT/EULAR (European League Against Rheu-matism) Autologous Stem Cell Transplanta-tion International Rheumatoid Arthritis Trial(3). However, this trial was suspended be-cause of insufficient patient accrual.

Systemic JIA is a heterogeneous form ofarthritis in childhood and represents 10-20%of all cases of JIA in the Caucasian popula-tions of Northern America and Europe. Upto 30% of patients will still have activedisease after 10 years, and morbidity withinthis group is high (18). Despite modern thera-pies, JIA remains a significant cause of mor-bidity in children because it does not usuallyrespond to anti-TNF agents.

Since 1997, HSCT has been applied asan experimental procedure to more than 50children with refractory JIA. In a retrospec-tive analysis of follow-up data on 34 chil-dren with JIA who were treated with ASCTat nine different European transplant cen-ters, the results showed that 18 of the 34patients (53%) achieved complete drug-freeremission at follow-up times of 12 to 60months. In 7 of these patients earlier treat-ment with anti-TNF had failed. Six of the 34patients (18%) showed a partial response(ranging from 30 to 70% improvement) and7 (21%) were resistant to ASCT. There were3 cases of transplant-related mortality (9%)and 2 of disease-related mortality (6%). Theseresults show that ASCT in severely ill pa-tients with JIA induces a drug-free remis-sion of the disease and a profound increasein general well-being in a substantial pro-portion of patients, but the procedure carriesa significant risk of mortality (19). Morerecently, this risk has been reduced by pre-treating with steroids the patients in whomthe disease is very active prior to transplan-tation. The European Bone Marrow Trans-

1585



www.bjournal.com.br

plantation Group plans to continue usingASCT in the treatment of JIA, particularlythe systemic onset form of the disease, withfludarabine in the conditioning regimen andprobably, in the future, MSC instead of HSC.Only 1 patient with RA was transplantedduring the Brazilian Cooperative Trial ofAutologous Hematopoietic Stem Cell Trans-plantation for AD. A 20-year-old femalepatient with the polyarticular form of JIAreceived a conditioning regime of CY (200mg/kg) and rabbit ATG (4.5 mg/kg) andautologous peripheral blood stem cells inSeptember/2005 at the Hospital Sírio Libanêsin São Paulo. Anti-CD20 monoclonal anti-body (Rituximab) was given (375 mg/m2

every 3 months) to prevent relapse and thepatient maintains complete remission after 1year of follow-up (20 and Massumoto C,personal communication).

Systemic lupus erythematosus

There is evidence that patients with SLE,a systemic AD, present BM dysfunction,with a marked reduction in the numbers ofCD34+ cells and a possibly reduced stem cellproliferation capability. The patients presentelevated levels of apoptosis in the CD34+

cell subpopulation and a reduced frequencyof colony-forming units when compared tocontrol groups. These abnormalities appearto be related to a defect in the microenviron-ment of the BM, contradicting the hypo-thesis that they are related to some primarydefect in the stem cells. During the post-transplantation period, a greatly reducednumber of apoptotic CD34+ cells are seenwhen compared with the pre-treatment pe-riod (21).

The EBMT and the EULAR registered53 cases of the use of HSCT in patients withSLE between 1995 and 2002. In the UnitedStates, a transplant center reported the use ofHSCT in 50 cases from 1997 to 2005 (21).Currently, research protocols use a condi-tioning protocol consisting of CY and G-

CSF for mobilization, and high doses of CYcombined with ATG, and in vitro CD34+ cellselection (21,22).

Besides the SLE patients submitted toHSCT reported in the EBMT/EULAR (21)and the American (22) studies, 32 otherisolated cases have been reported. Therewere 22 cases of clinical remission. About30% of the patients who had remission of thedisease presented a relapse (23).

With this disease, the longer the post-transplantation period, the greater the risk ofrelapse, which is associated with low levelof anti-dsDNA antibodies prior to HSCT.According to the EBMT/EULAR studies,there was no difference in the frequency ofrelapse with selection of CD34+ cells or witha more intense conditioning regime. Theadverse effects of the treatment are not se-vere and the activity of the disease can becontrolled with therapies to which the pa-tient was previously resistant (21,22).

There are many examples of clinical im-provement in patients with SLE submitted toAHSCT or to treatment with high doses ofCY (24). However, new inflammatory andautoimmune processes have occurred in pa-tients submitted to HSCT (21) and in pa-tients treated only with CY without infusionof stem cells (24). This probably occurs asthe result of the deletion of regulatory T cells(21).

In Brazil, the first four cases of SLEsubmitted to AHSCT in a multicenter coop-erative trial were reported in 2003 (1). Thepatients received high doses of CY and ATG,which were both administrated before andafter cell infusion. The use of ATG immedi-ately after infusion was intended to comple-ment the in vivo T cell deletion since in theprotocol the infused cells did not suffer invitro selection. Three patients presented se-vere kidney failure and 1 patient died. The 3surviving patients showed remission of kid-ney disease after transplantation, but 1 pa-tient relapsed thereafter and showed a de-cline in renal function (Scheinberg M, per-

1586


S.B. Rosa et al.

www.bjournal.com.br

sonal communication). Four other patientswere included in the clinical trial, 1 patientcould not have his stem cells mobilized andreturned to conventional immunosuppres-sion and 3 patients died from transplant com-plications (acute renal failure and septice-mia) (20).

Several studies in the international litera-ture have shown that AHSCT is efficient inachieving remission of disease in patientswith resistant SLE. Transplantation is ableto change the behavior of severe diseases,making them more benign and responsive totherapy. It has been suggested that the re-ported higher rates of mortality in Brazil andother countries (25) can be controlled byappropriate patient selection, the choice of aless aggressive conditioning regime and theacquisition of more experience in managingspecific complications associated with theprocedure (21).

Multiple sclerosis

Multiple sclerosis is an organ-specificAD mediated by T cells triggered againststructural components of myelin in the cen-tral nervous system (CNS). Subsequent toinflammation in the CNS, demyelinizationand loss of axons may occur, resulting ininterruption of the electrical signal. MostMS patients present episodic relapse andimprovement, known as relapsing-remittingMS, followed by a phase called secondaryprogressive MS. There is yet another form ofMS known as primary progressive MS, whichis generally resistant to conventional thera-pies (26).

Available treatments for MS are not cura-tive. They are able to reduce inflammation inthe CNS and to delay the advance of thedisease, but disease control is frequentlyunsatisfactory. The use of stem cells in thetreatment of MS is based on the immunosup-pressor and immunomodulatory effects ofAHSCT, which may favor the immunologi-cal balance (26). Furthermore, the multi-

focal nature of MS makes the injection ofstem cells into each affected site impracti-cable, which means that the cells need to beattracted to the pathological areas. The intra-venous administration of stem cells may bean alternative in MS and other neuroinflam-matory conditions, in which there is perme-ability of the hematoencephalic blood brainbarrier in the inflammatory areas. Moreover,the discovery that stem cells are capable ofreaching the CNS and of transdifferentiatingor acquiring oligodendrocyte and possiblyneuronal properties, suggests that they maybe able to act in re-myelinization and neuronrepair.

The first reported AHSCT in patientswith MS was performed in 1995 (26). ByNovember 2002, MS was the AD with thelargest number of reports of AHSCT. Morethan 200 patients were submitted to AHSCTspecifically for the treatment of MS (3).Table 2 lists the clinical trials of HSCT forMS. Practically all the patients were in theprogressive phase of MS, and most of themwere in the secondary progressive MS phase.These patients had not responded to previ-ous conventional treatments and presentedsevere functional incapacity. There is novariation in the results from different trans-plant centers (27). AHSCT significantly re-duced the inflammation in the CNS, as shownby magnetic resonance imaging, and en-hanced the results obtained with conven-tional therapies (26). The most common ad-verse effect was the occurrence of infection,which affected more than 50% of the pa-tients in the clinical trial of Fassas and col-leagues (27). The mortality rate associatedwith transplantation is high (5-10%) whencompared with that of conventional treat-ment (0%), although it is believed that witha more precise patient selection procedurethis rate could be reduced. The morbidityand mortality rates seem to be related to theintensity of T cell deletion and to the condi-tioning regime, as well as to advanced ageand severe functional incapacity of the pa-

1587



www.bjournal.com.br

tients (26). There is a need to determine whatdegree of risk is acceptable in patients with adisease that, although severe and rarely cur-able, has a low death rate.

Some clinical studies have reported thepersistence of cerebral atrophy after AHSCTfor MS. However, it is still not possible toknow whether or not AHSCT has any effecton the advance of atrophy because the pa-tients involved in these studies presented asevere and rapidly advancing form of MS. Inthis situation, cerebral atrophy might be tak-ing place at an accelerated and cumulativepace and, moreover, a more active phase ofthe disease may occur during pre-treatment,mainly during cell mobilization. Accord-

ingly, the loss of brain tissue could be a long-term consequence of previous activity of thedisease. Furthermore, there is the possibilityof pseudoatrophy, which occurs in responseto the intense suppression of the diseaseactivity induced by AHSCT (28).

AHSCT appears not to yield good resultsin patients whose clinical progression in-volves axon degeneration. Thus, the use ofAHSCT in the early stages of MS wouldappear to be more suitable for patients with alow level of functional incapacity (26).

Matrix metalloproteinase-9 (MMP-9), amarker of MS pathogenesis, is regulated bythe tissue inhibitor of MMP-1. The serumand expression level of mRNA of MMP-9

Table 2. Protocols of transplantation and results of clinical studies based on the use of autologous peripheral blood hematopoietic stem celltransplantation in multiple sclerosis.

Number of Number of deaths Results and Conclusions Referencespatients

85 Total: 7 · Better results in patients with SPMS and less than 40 years old 27PBHSC: 79 5: related with · Improvement of neurological symptoms in 18 patients (21%) and reduction of activityBM: 6 transplantation; detected by MRI

2: normal · Absence of significant difference in results: duration of the disease pre-transplantation,progression of the conditioning regime, ex vivo deletion of lymphocytedisease · Stem cell transplantation showed better results than IFN-ß or placebo

35 Total: 2 · Reduction of activity detectable by MRI in spite of permanence of brain atrophy 291: related with · Development of post-transplantation AD (autoimmune thyroid and inhibition of factor VIII)transplantation; · Greater benefits in patients with SPMS and RRMS, than patients with PPMS1: delayed effect

15 0 · Stability or improvement in 12 patients 30· Progression of MS: 2 patients (1 year post-transplantation)· Only 2 patients had relapse· Reduction of activity detectable by MRI in spite of permanence of brain atrophy

26 0 · Clinical improvement in 1 patient, stable condition in 13 patients and deterioration in 317 patients

· MRI: new lesions in 4 patients· Probability of absence of progression of the disease: greater in patients with SPMS than

in patients with PPMS

19 0 · Maintenance treatment was not administered for an average period of 36 months post-HSCT 32· MRI: only 1 patient presented new lesion in 4.5 years· Improvement in quality of life· Survival free of disease advancement: 95 ± 5% in 6 years post-HSCT· Survival free of disease activity: 64% in 4.5 years post-HSCT

The studies cited derive from a review of the literature and give a general view of the different outcomes after therapy. AD = autoimmune disease; BM= bone marrow; HSCT = hematopoietic stem cell transplantation; IFN = interferon; MRI = magnetic resonance imaging; MS = multiple sclerosis;PBHSC = peripheral blood hematopoietic stem cells; PPMS = primary progressive multiple sclerosis; RRMS = relapsing-remitting multiple sclerosis;SPMS = secondary progressive multiple sclerosis.

1588


S.B. Rosa et al.

www.bjournal.com.br

and of tissue inhibitor of MMP-1 were ana-lyzed in peripheral mononuclear cells from14 patients with MS after AHSCT. The re-sults indicated inhibition of MMP-9 activ-ity, which is in accordance with clinicalcondition and reduction of disease activity(33).

The mechanism by which AHSCT af-fects the course of MS is not known. Theimmediate effect appears to be the eradica-tion of auto-reactive clones. Patients treatedwith AHSCT show a low T CD4+ cell count(33), and, as a consequence, the inflamma-tory process is reduced and there is a pos-sible clinical improvement. Besides this im-mediate effect, the infusion of stem cellsafter high doses of immunosuppression ap-pears to give rise to a “new” tolerance sys-tem (34). Another hypothesis to explain theimprovement of patients is the capacity ofstem cells to transdifferentiate into glial cellsand neuronal precursors, contributing to therepair of damaged nervous tissue. Thus,AHSCT can lead to prolonged periods ofstabilization of MS or change the degree ofseverity in the course of the disease.

An initiative of the EBMT/EULAR, theAutologous Stem Cell Transplantation In-ternational Multiple Sclerosis Trial intendsto compare AHSCT with conventional treat-ment, and so determine the efficacy andtoxicity related to stem cell therapy in pro-gressive MS (3). Another controlled clinicaltrial (MIST), created by R. Burt in Chicagowith the cooperation of some Brazilian cen-ters, compared AHSCT with conventionaltherapy in the earlier inflammatory phase ofinterferon-refractory diseases (relapsing-re-mitting).

Under the Brazilian cooperative trial ofHSCT for AD, 41 patients with progressive(primary or secondary) MS with a score ≤7.0on the Expanded Disability Status Scale weretransplanted from June/2001 through Au-gust/2006 at several centers, mainly in theUniversity Hospital of the Ribeirão PretoMedical School and the Hospital Israelita

Albert Einstein in São Paulo. Initially, 21patients were conditioned with 1,3-bis[2-chloroethyl]-1-nitrosourea, etoposide, ara-cytin, melphalan, and horse ATG, and 3patients died from transplant-related com-plications. Beginning in 2004, following achange in the conditioning regime to CYplus ATG, there were no deaths among 20patients transplanted and there was no dif-ference in the rate of response (stoppingprogression) between the two groups (be-fore and after the change in the conditioningregime). Eight patients transplanted out ofprotocol (excessively high Expanded Dis-ability Status Scale or stronger immunosup-pression) either died or showed neurologicalprogression (Hamerschlak N and VoltarelliJ, personal communication).

Systemic sclerosis

Systemic sclerosis or scleroderma is char-acterized by predominant T cell activation,autoantibody production (anti-scleroderma-70) and cytokine secretion, leading to exces-sive fibrosis throughout the body. Severeforms of the disease give rise to high levelsof morbidity and mortality (estimated at 40-50% in 5 years), secondary to lung, cardiacand kidney involvement. Conventional thera-pies apparently are not efficient in prevent-ing the advance of the disease and reversionof the fibrosis (35).

In November 2003, 83 cases of patientswith SS submitted to stem cell transplanta-tion were registered in Europe and the UnitedStates (35). The therapeutic regimes usedfor the treatment of SS with ASCT regis-tered in EBMT/EULAR were similar. Forcell mobilization, the use of CY and G-CSFis preferred to that of G-CSF only. The se-lection of CD34+ cells prior to re-infusionwas performed in 87% of patients. For con-ditioning, CY was applied in 90% of cases,either as a monotherapy or in combinationwith ATG (36).

Since 1996, good results have been ob-

1589



www.bjournal.com.br

tained in SS after HSCT. In a study pub-lished in 2006, which included 6 patientswith SS, 1 of them with associated SLE,improvement was reported in dermatologicsclerosis in all patients (37).

In a study by Farge and colleagues (36),57 patients with SS were treated with HSCTin the period from 1996 to 2002 and fol-lowed up for more than 6 months. Completeremission was observed in 14 patients, andpartial remission in 32 patients, for a periodof over 3 years. Though some patients ex-hibited active disease following HSCT, theywere found to be in better clinical conditionthan prior to treatment.

However, not all the reported outcomesare favorable. In a French multicenter studyinvolving 11 patients, while a complete orpartial response was observed in 8 patients, 5of these patients showed reactivation of thedisease. Nevertheless, according to the au-thors, AHSCT is able to extend the short lifeexpectancy of patients with severe SS (36).

Toxicity risk factors were not totally an-alyzed due to the small number of patientsand to the variety of protocols applied. Thera-py with high doses of immunosuppressorsappears to be less tolerated by patients withlung and heart damage due to the cardiotox-icity of CY. A careful selection of patients,close monitoring during treatment andchanges in the transplantation protocol shouldreduce the number of deaths (35). A condi-tioning regime using thiotepa and low dosesof CY seems to minimize the cardiotoxicityinduced by CY (38), and it is recommendedthat TBI in the absence of lung protection beavoided.

Supervised by the EBMT and EULAR, acontrolled and randomized phase III study isbeing organized, called Autologous StemCell Transplantation International Sclero-dermal Trial (3). In the United States, asimilar randomized phase III study, calledScleroderma: Cyclophosphamide or Trans-plantation, is being planned. The aim ofthese studies is to investigate the effects of

intensive immunosuppression followed byHSCT versus conventional chemotherapywith CY in patients with scleroderma inorder to establish a safer and more effectiveprotocol (35).

In the Brazilian cooperative trial ofAHSCT for AD, seven patients with pro-gressive and refractory SS were treated (20).Three patients were mobilized but not trans-planted (2 improved and 1 died from infec-tion). One patient with overlap syndrome(SLE plus SS) died from disease reactiva-tion (pulmonary and cerebral vasculitis) be-fore receiving HSCT. Three patients im-proved significantly after transplantation(softening of the skin and stabilization ofpulmonary function), but 1 of them died ofpulmonary infection more than 2 years aftertransplantation. After this report, 2 otherpatients were transplanted at the Universityof São Paulo Hospital in Ribeirão Preto with-out early complications and with rapid im-provement of skin lesions.

Autoimmune cytopenias

Autoimmune thrombocytopenic purpura(AITP) is a disease in which platelets aresensitized by anti-platelet antibodies or cir-culating immune complexes, provoking theearly removal of these cells from the circula-tion. This process results in thrombocytope-nia and bleeding. About one third of patientswith AITP do not respond well to conven-tional therapies. High doses of immunosup-pressors may lead to remission of the dis-ease; however, this involves risks related tomyelosuppression. HSCT aims to acceleratereestablishment of the hematological param-eters and concomitantly reduce the numberof autoimmune cells in the organism (39).Several sporadic case reports of HSCT forautoimmune cytopenias have been published,but only two studies have been reported withmoderate numbers of patients.

A study described the clinical history ofpatients registered in the EBMT who had

1590


S.B. Rosa et al.

www.bjournal.com.br

received HSCT to treat severe refractoryautoimmune cytopenia. In 12 patients withAITP submitted to autologous transplanta-tion the responses were highly diverse, vary-ing from a transient response to continuousremission or even death related to transplan-tation (40). In the US, of 14 patients withchronic refractory AITP submitted to highdose cyclophosphamide (200 mg/kg) fol-lowed by AHSCT, 6 achieved durable com-plete responses and 2 obtained durable par-tial responses (41). The study concluded thatthe infusion of HSCs had accelerated thehematological reestablishment. Since clini-cal improvement was not associated withquantification of anti-GPIb or anti-GPIIIaantibodies, it is likely that other platelet an-tigens were involved in the AITP. The re-sponses were not associated with the num-ber of CD3+ cells infused into the patients,although the deletion of T lymphocytes mayhave prevented the re-infusion of auto-reac-tive T cells (39).

Clinical trials involving larger numbersof patients could provide more reliable in-formation on the importance of lymphocytedeletion and immunogenetic characteriza-tion of patients that are responsive or non-responsive to treatment. It would then bepossible to compare different treatment pro-tocols: a) therapy based only on high dosesof CY; b) therapy using high doses of CYcombined with stem cell transplantation, andc) the latter procedure used in associationwith agents inducing the maintenance ofpost-transplantation tolerance (39).

Autoimmune hemolytic anemia (AIHA)is characterized by the early destruction oferythrocytes due to the fixation of immuno-globulin or complement protein on the mem-brane surface. The initial symptoms can re-sult from anemia caused by hemolysis, thesecondary effects of the hemolytic condi-tion, or the primary disease which caused theAIHA. There are a few reports in the scien-tific literature of cases in which stem celltransplantation has been used for the treat-

ment of AIHA (40,42) and the data suggestthat immunosuppressive therapies should beused post-transplantation in this disease.

Despite the incipient data, stem cell trans-plantation offers a new therapeutic possibil-ity for patients with AIHA.

Diabetes mellitus

In recent years, there has been a rapidgrowth in the number of cases of diabetesthroughout the world. This epidemic affectsapproximately 6-8% of the world populationand the number of newly diagnosed patientsincreases yearly (43). Of these, approxi-mately 10% are type I diabetes, insulin-dependent diabetes mellitus, an AD causedby the progressive destruction of the insulin-secreting pancreatic ß-cells in the islets ofLangerhans (4,44) which regulate bloodsugar levels by secretion of insulin. Recentclinical data suggest that the disease couldbe cured if an adequate supply of new ß-cellswere made available. Hence, one goal ofpancreatic developmental biology is to un-derstand how endogenous ß-cells are made,with the hope of producing them exogenously(44).

Pancreatic islet cell transplantation is anattractive treatment of type I diabetes (45).Clinical islet transplantation trials based oncadaveric allogeneic islets have demonstratedthat it is indeed possible to restore near-physiological insulin secretion capacity intype I diabetic patients through transplanta-tion of insulin-producing cells (46). Thereare a number of difficulties associated withislet allotransplantation such as problemsrelated to alloimmunity, autoimmunity, andthe need for larger islet cell masses. Employ-ment of animal islets and stem cells as alter-native sources of insulin production will beconsidered in order to deal with the problemof human tissue shortage (45). Lifelong im-munosuppressive therapy and inadequatesources of transplantable islets have limitedthe benefits of islet transplantation to less

1591



www.bjournal.com.br

than 0.5% of type I diabetics. Whereas thepotential risk of infection by endogenousanimal viruses limits the uses of islet xeno-transplantation, deriving islets from stemcells seems to be able to overcome the cur-rent problems of islet shortages and immunecompatibility (4).

The fact that BM contains HSCs andMSCs, which are capable of differentiatinginto a number of different kinds of cells suchas pancreatic ß-cells, has, in recent years,attracted the attention of researchers to thestudy of stem cells in the treatment of type Idiabetes. However, there is some contro-versy regarding the results obtained withboth ESCs and ASCs, in relation to theirpotential for in vitro secretion of insulin andin vivo normalization of hyperglycemia (4).Stem cells from the spleen have been dem-onstrated to home to the pancreas wherethey mature into fully functional islet cellsresponsible for restoring normoglycemia(47).

Transplantation of ESC-derived insulin-producing cells has reversed diabetes in ani-mals, indicating that these cells synthesizeand release insulin. Experiments on diabeticmice with ß-cells totally destroyed by strep-tozocin showed that after BMT blood glu-cose was normal and survival was longer(4).

A recent study demonstrated the poten-tial use of primary cultures of adult humanliver cells as pancreatic progenitors, show-ing the plasticity of immature liver cells. Thepossibility of using liver as a pancreaticprogenitor tissue has been demonstrated bothin vivo and in vitro in Xenopus mice andhumans. The use of adult human liver cellsfor generating functional insulin-producingtissue may pave the way to autologous im-plantations, thus allowing the diabetic pa-tient to be the donor of his or her owninsulin-producing tissue (43).

Stem cells offer the best chance of achiev-ing this goal. Controversial results have beenpresented concerning the existence and na-

ture of pancreatic islet stem or precursorcells (46). There is also controversy regard-ing the results obtained with ESCs and ASCsduring in vitro secretion of insulin and invivo normalization of hyperglycemia. Re-search into ESCs is thought to have muchgreater developmental potential than researchinto ASCs; however it is still in the basicresearch phase. Existing ESC lines are notbelieved to be identical or ideal for generat-ing islets or ß-cells and additional ESC lineshave to be established (4).

HSC chimerism achieved through BMTmay affect type I diabetes in two ways: first,by inducing tolerance to pancreas and isletcell transplants, and second by reversing theautoimmune process prior to the develop-ment of terminal complications (48).

These studies show the possibility ofBMT as a therapeutic approach for ß-cellreplacement (4). However, allogeneic BMTdoes have limitations including the morbid-ity associated with lethal conditioning, graft-versus-host disease, and failure of engraft-ment. Currently, the morbidity and mortalityassociated with lethal conditioning couldnot be justified for tolerance induction orinterruption of the autoimmune state in typeI diabetes (48). While immune destruction isa problem in BMT, transplanting splenicmesenchymal cells has yielded promisingresults in both pancreatic ß-cell regenerationand immune destruction prevention (4).

Significant advances have occurred inthe treatment of type I diabetes during thepast century, but BMT is the only methodcapable of interrupting and reversing theautoimmune process prior to the develop-ment of terminal complications. When limi-tations associated with conventional BMTare overcome, BMT may become a thera-peutic option in the treatment of autoim-mune diabetes and tolerance induction. Tech-niques that promise to facilitate the use ofBMT are currently being studied, includingthe development of partial conditioning strat-egies, BM graft engineering to enrich HSCs

1592


S.B. Rosa et al.

www.bjournal.com.br

and cell populations that facilitate engraft-ment and approaches to limit graft-versus-host disease. The use of various stem celltypes as sources for ß-cell regeneration isalso important, as recently reviewed (49).The end result will be safer BMT, therebyallowing its use in nonmalignant settingssuch as AD and tolerance induction in solidorgan and cell transplantation (48).

From December 2003 through July 200615 patients with early onset type I diabetesmellitus (less than 6 weeks from diagnosis)received AHSCT at the University Hospitalof the School of Medicine of Ribeirão Preto,using CY (200 mg/kg) and rabbit ATG (4.5mg/kg) as a conditioning regime (50). Of 14patients without previous ketoacidosis andsteroids for conditioning, 12 became insu-lin-free shortly after transplantation, 2 after1 year from transplantation and 1 patientrelapsed after a viral infection.

Amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is afatal disease characterized by the progres-sive death of central and peripheral motorneurons, which results in severe motor dys-function (5,51). In a few cases, between 5and 10%, the origin is hereditary, apparentlycaused by a mutation of the superoxide dis-mutase gene (52), but in most cases (spo-radic ALS) the cause of the disease in un-known. Autoimmune and inflammatory ab-normalities may influence the progressionof the disease but ALS is not a typical AD.The therapeutic strategy used in cases ofALS involves attempting to prevent neu-ronal loss and to stimulate cell regeneration.At present, the combined therapies designedto halt the advance of the disease, which useneuroprotective agents and drugs, have astatistically significant but small influenceon patient survival. However, these thera-pies are incapable of restoring the lost motorfunction. In ALS, the therapeutic target is toimprove the interaction between motor neu-

rons and non-neuronal cells, mainly glialand microglial cells. Stem cell therapy is apromising strategy for restoring neuromotorfunction and/or preventing motor-neurondegeneration in cases of ALS (5).

Until a few years ago, it was believed thatneuronal tissue was unchangeable. How-ever, now it is known that cell replacementoccurs in adulthood in some specific regionsof the brain. Difficulty in obtaining adultneural stem cells has lead to the use of UCBand BM as alternative sources of stem cellsin the therapy for ALS. These cells can beexpanded ex vivo, inducing differentiationinto the desired cellular type and then trans-planted (5).

Transdifferentiation of human HSCs fromUCB to neurons has been tested in vitro.These cells are differentiated into neuralcells and, sequentially, into astrocytes in asuitable microenvironment. There are re-ports showing differentiation of ESCs intomotor neurons in mice. However, the capac-ity of transdifferentiation is controversialand other biological explanations have beensuggested, particularly cell fusion. Cogleand colleagues (6) showed the differentia-tion of human HSCs into neurons, astrocytesand microglial cells in the absence of cellfusion.

In another study, after infusion of humanESCs into the cephalorachidian liquid (CRL)of SOD1 mice, stem cells migrated towardsthe damaged area of the spinal cord, andabout 6% of these injected cells gave rise tonew neurons and the treated mice showedreversal of paralysis (52). According toSvendsen and Langston (51), these resultsare mainly due to the differentiation intoglial cells and to the production of neuropro-tective trophic factors, and not to the directreplacement of motor neurons.

Given the limitation of cellular transdif-ferentiation, the tendency of neural stem cellgrafts to differentiate into glial cell linesafter implantation in the spinal cord and thedifficulty in transdifferentiating human blood

1593



www.bjournal.com.br

cells into neuroglial cells in vitro, the thera-py based on stem cells in ALS is still a longway from becoming routine. Nevertheless,the therapeutic needs of these patients haveled to the clinical studies, despite the ab-sence of conclusive evidence. In a clinicalstudy, CD34+ cells purified from peripheralblood were injected into 3 patients with ALS.After 12 months, while none of the patientsshowed any severe adverse effects, therewas no apparent clinical benefit. However,other studies have shown more promisingresults. Expanded MSCs were injected intothe CRL of 7 patients and 3 months aftertransplantation 4 of them showed a positiveclinical response (5).

The aim of applying stem cells in casesof ALS is to achieve differentiation of thesecells into motor neurons and the establish-ment of appropriate functional connections.Stem cells may also act through the deliveryof trophic and growth factors, giving rise toa favorable environment for the survival ofneural cells and acting as protective agentsagainst neurotoxic substances (5).

Most clinical studies involving the use ofcell therapy in cases of ALS have employedthe injection of autologous stem cells orESCs directly into the CNS. Janson andcolleagues (53), after trying the technique inprimates, injected selected HSCs (CD34+

cells) into the spinal intrathecal space of 3patients. The authors observed partial ormild effects on neurological function andthe absence of complications. In anotherstudy carried out at the University of Turin,Italy (54), 7 patients with severe impairmentof the lower limbs but no respiratory symp-toms, received ex vivo expansion of autolog-ous MSCs from BM directly into the surgi-cally exposed spinal cord (T7-T9 level). Mostof the patients only presented slight andreversible side effects (intercostal pain ordysesthesia), a result confirmed after a 2-year follow-up (55). Deceleration of the lossof muscular strength in the legs was ob-served 3 months after transplantation in 4

patients together with a slight improvementin 2 patients (54). Additionally, there wasalso a deceleration in the decline of respira-tory function in 4 patients after 2 years.These effects were correlated with the num-ber of injected cells (55). At a scientificevent (56), a report was presented about theuse of stem cells extracted from 4- to 8-weekembryos that were injected into 61 patientswith ALS on 1 to 4 occasions. Improvementof neurological function was reported in 34%of the patients after 2 months and, subse-quently, in those patients who received re-peated injections. The findings showed thatthe outcome was better in cases where thedisease was at an earlier stage, but there areno data regarding survival. A researcher fromMontevideo, Uruguay, treated 12 ALS pa-tients with HSCs collected from peripheralblood, suspended in CRL and directly in-jected into the spinal cord. The results werequite encouraging, since neurological func-tion improved in 3 patients and was stabi-lized in 9 (57).

There are few reports of the use of HSCT,followed by high dose immunosuppressionin ALS. A report was sent to the EuropeanRegistry of HSCT for AD (EBMT/EULAR,based in Basle, Switzerland) of 2 patientssubmitted to AHSCT in France (Tyndall A,personal communication). Despite transitoryimprovement over a period of 3 months in 1of the patients, both died about 7 monthsafter transplantation. In the US, a group ofscientists from Baylor College of Medicine,Houston, TX, carried out a pilot study in-volving 6 patients at intermediate stages ofthe disease, who received allogeneic BMTfrom their HLA-identical siblings (58). Allthe patients underwent donor cell graftingand 3 patients showed no benefits from theprocedure, with 2 of them dying and thedisease continuing to advance in the other.Nevertheless, the 3 remaining patientsshowed a reduction in the advance of thedisease. Autopsy of 1 of the patients re-vealed large quantities of donor DNA in

1594


S.B. Rosa et al.

www.bjournal.com.br

various regions of the spinal cord and brain,demonstrating that allogeneic BM cells werecapable of crossing the blood brain barrierand incorporating themselves into the ner-vous tissue.

There is evidence that the inflammatoryprocess is a key factor in the pathogenesis ofALS (59) and that high dose immunosup-pression with stem cell transplantation mightchange the natural history of the disease(60). Based on this evidence, AHSCT forALS started in Brazil in December 2004 atthe University Hospital of the School ofMedicine of Ribeirão Preto. Two patientswere transplanted using a triple immunosup-pressive regimen (CY, ATG, and fludara-bine). The first one with the bulbar form diedat D+103 from pulmonary embolism and thesecond is still alive 28 months after trans-plantation. A more extensive experience ofAHSCT was obtained at the Salvador Hos-pital in Bahia where 15 patients were trans-planted using the same conditioning regime.There were 3 transplant-related deaths, 1progression-related death and a sudden deathunrelated to transplantation or progression.Ten patients are alive after a mean follow-upof 133 days (1-540) (Pallotta R, personalcommunication). Both protocols were haltedby the national institutional review board(CONEP/Ministry of Health) pending refor-mulation.

Conclusion

Stem cell transplantation has been rec-ommended for use in the treatment of ADbecause it provides immunosuppressive andimmunomodulatory effects. Stem cell trans-plantation can destroy the defective immunesystem, renew the lymphatic system, reducethe disease activity, and lead to long-termremission of AD (27). AD patients who wereonce resistant to conventional therapy, fol-lowing transplantation, have become sensi-tive to the same conventional therapy, high-lighting the immunomodulatory properties

of stem cell transplantation.Most clinical studies based on stem cell

therapy in AD patients use AHSCT mobi-lized from BM to peripheral blood. There isno significant difference in the transplanta-tion protocols used for different diseases.The results shown in this review range fromcases of complete or partial remission, re-lapse or stabilization of disease to casesinvolving the advance of the disease anddeath. Patient selection appears to directlyinfluence the results of transplantation.Choosing patients in the initial stages of thedisease appears to be safer and more effec-tive. However, this procedure involves therisk of exposing patients who may respondwell to conventional therapy to new non-standard treatment methods (21). It shouldbe pointed out that most of the proceduresreviewed here were performed in patientsthat failed to respond to conventional treat-ment therapies. In such patients, the use ofstem cells, although still being an experi-mental procedure, provides a new chance toenhance the quality of life.

There is some controversy regarding theuse of high doses of chemotherapy in theabsence of stem cell infusion, which, in somecases, has led to satisfactory results, sug-gesting that immunosuppression may be thereal cause of the improvement seen in thepatients, and not the stem cells (26). Also,the total number of patients assessed in somestudies is quite small since they representcase reports and small uncontrolled case-series. In addition, the individuals representhighly diverse genetic backgrounds, and onlyrandomized clinical trials following stand-ard procedures will allow us to reach mean-ingful conclusions. Furthermore, there is theneed for studies which will evaluate the useof maintenance therapies after transplanta-tion, define patient selection protocols andassess stem cell mobilization in the treat-ment of AD.

Patient relapse is more common in auto-logous (30%) than in allogeneic (5%) trans-

1595



www.bjournal.com.br

plantation, with the latter also producinglonger-lasting results (26). Relapse could becaused by contamination of the graft withautoimmune cells, the incapacity of the con-ditioning regime to destroy the immune cellsresponsible for the disease or the presence ofthe defect in the stem cell expansion (14).The contamination of grafts by auto-reactivelymphocytes can be controlled using purecell lines expanded in the laboratory, whichwould also increase the number of stem cellsavailable for the patient (26). The efficacy ofan allogeneic transplantation is mainly theresult of the change in the genetic suscepti-bility of the host to the disease, and alsoinvolves the graft-versus-disease effect. Cur-rently, this procedure is not frequently useddue to its toxicity and the need for HLAcompatibility (9,14).

It is known that ESCs have a greatercapacity for differentiation and less immu-nogenetic potential than ASCs. Neverthe-less, ESCs could be used as an alternativewhen there is a high risk of immunologicalrejection with ASC therapy, as in the case ofallogeneic transplantation, or in cases wherethere is relapse as a consequence of thepatient genotype, as occurs in autologoustransplantation. However, the use of ESCsremains limited and far from being a thera-peutic alternative for such patients due to therisk of the development of teratomas and theethical issues involving the use of this proce-dure.

MSCs represent a promising therapy forAD. These cells present immunosuppres-sive properties and there are reports suggest-ing the low level of expression of HLA

molecules in MSCs. It is believed that thetherapy mediated by mesenchymal cells maypermit the secretion of immunosuppressorfactors and may repair the tissue destroyedby the chronic inflammatory process.

A mixed approach involving the combi-nation of genetic transference with cellulartherapy is able to increase the therapeuticpotency of stem cell transplantation. It per-mits the correction of genetic anomalies andhelps in the control of disease by increasingthe expression of regulatory elements (21).In neurodegenerative diseases, neural stemcells may remain in the quiescent stage dueto the unfavorable microenvironment causedby disease. It is believed that geneticallymodified stem cells can stimulate the prolif-eration of endogenous stem cells and so leadto repair of the damaged neural region (5).

An interesting strategy would be to labelthe infused stem cells for later evaluation oftheir proliferation, differentiation, migration,and homing. Cellular labeling, such as thatachieved by Cogle and colleagues (6), makesit possible to expand the knowledge avail-able on the activity and biology of stemcells, to evaluate the effectiveness of trans-plantation and to develop better protocolsfor the application of stem cells to eachimmunological disease.

Stem cell-based therapy offers the possi-bility of developing new treatments in casesof AD. Several reports have analyzed thepotential benefits of stem cell transplanta-tion in these diseases. It is hoped that newstudies currently underway will show goodresults for the treatment of AD in the nearfuture.

References

1. Voltarelli JC, Stracieri ABPL, Oliveira MCB, Paton EJA, Dantas M.Transplante autólogo de células tronco hematopoéticas para nefritelúpica: resultados brasileiros iniciais. J Bras Nefrol 2003; 25: 65-72.

2. Snowden JA, Kearney P, Kearney A, Cooley HM, Grigg A, JacobsP, et al. Long-term outcome of autoimmune disease following allo-geneic bone marrow transplantation. Arthritis Rheum 1998; 41: 453-459.

3. Hough RE, Snowden JA, Wulffraat NM. Haemopoietic stem celltransplantation in autoimmune diseases: a European perspective.Br J Haematol 2005; 128: 432-459.

4. Miszta-Lane H, Mirbolooki M, James Shapiro AM, Lakey JR. Stemcell sources for clinical islet transplantation in type 1 diabetes:embryonic and adult stem cells. Med Hypotheses 2006; 67: 909-913.

1596


S.B. Rosa et al.

www.bjournal.com.br

5. Silani V, Cova L, Corbo M, Ciammola A, Polli E. Stem-cell therapyfor amyotrophic lateral sclerosis. Lancet 2004; 364: 200-202.

6. Cogle CR, Yachnis AT, Laywell ED, Zander DS, Wingard JR,Steindler DA, et al. Bone marrow transdifferentiation in brain aftertransplantation: a retrospective study. Lancet 2004; 363: 1432-1437.

7. Wulffraat M, de Kleer I, Brinkman D, ten Cate R, van der Net JJ,Rijkers GT, et al. Autologous stem cell transplantation for refractoryjuvenile idiopathic arthritis: current results and perspectives. Trans-plant Proc 2002; 34: 2925-2926.

8. Snowden JA, Passweg J, Moore JJ, Milliken S, Cannell P, Van LaarJ, et al. Autologous hemopoietic stem cell transplantation in severerheumatoid arthritis: a report from the EBMT and ABMTR. JRheumatol 2004; 31: 482-488.

9. Bingham SJ, Moore JJ. Stem cell transplantation for autoimmunedisorders. Rheumatoid arthritis. Best Pract Res Clin Haematol 2004;17: 263-276.

10. Weyand CM, Goronzy JJ. Stem cell aging and autoimmunity inrheumatoid arthritis. Trends Mol Med 2004; 10: 426-433.

11. Burt RK, Oyama Y, Verda L, Quigley K, Brush M, Yaung K, et al.Induction of remission of severe and refractory rheumatoid arthritisby allogeneic mixed chimerism. Arthritis Rheum 2004; 50: 2466-2470.

12. Breban M, Dougados M, Picard F, Zompi S, Marolleau JP, BocaccioC, et al. Intensified-dose (4 gm/m2) cyclophosphamide and granulo-cyte colony-stimulating factor administration for hematopoietic stemcell mobilization in refractory rheumatoid arthritis. Arthritis Rheum1999; 42: 2275-2280.

13. Snowden JA, Biggs JC, Milliken ST, Fuller A, Brooks PM. A phase I/II dose escalation study of intensified cyclophosphamide and auto-logous blood stem cell rescue in severe, active rheumatoid arthritis.Arthritis Rheum 1999; 42: 2286-2292.

14. Burt RK, Georganas C, Schroeder J, Traynor A, Stefka J, SchueningF, et al. Autologous hematopoietic stem cell transplantation in re-fractory rheumatoid arthritis: sustained response in two of four pa-tients. Arthritis Rheum 1999; 42: 2281-2285.

15. Brodsky RA, Petri M, Smith BD, Seifter EJ, Spivak JL, Styler M, et al.Immunoablative high-dose cyclophosphamide without stem-cell res-cue for refractory, severe autoimmune disease. Ann Intern Med1998; 129: 1031-1035.

16. Moore J, Brooks P, Milliken S, Biggs J, Ma D, Handel M, et al. A pilotrandomized trial comparing CD34-selected versus unmanipulatedhemopoietic stem cell transplantation for severe, refractory rheuma-toid arthritis. Arthritis Rheum 2002; 46: 2301-2309.

17. McCool G, Hohsaka H, Wicks I. High-dose chemotherapy and syn-geneic hemopoietic stem-cell transplantation for severe, seronega-tive rheumatoid arthritis. Ann Intern Med 1999; 131: 507-509.

18. Woo P. Systemic juvenile idiopathic arthritis: diagnosis, manage-ment, and outcome. Nat Clin Pract Rheumatol 2006; 2: 28-34.

19. de Kleer IM, Brinkman DM, Ferster A, Abinun M, Quartier P, Van DerNet J, et al. Autologous stem cell transplantation for refractoryjuvenile idiopathic arthritis: analysis of clinical effects, mortality, andtransplant related morbidity. Ann Rheum Dis 2004; 63: 1318-1326.

20. Voltarelli JC, Stracieri ABPL, Oliveira MCB, Godoi DF, Moraes DA,Pieroni F. Transplante de células tronco hematopoéticas em doen-ças reumáticas. Parte 2: Experiência brasileira e perspectivas futu-ras. Rev Bras Reumatol 2005; 45: 301-312.

21. Jayne D, Passweg J, Marmont A, Farge D, Zhao X, Arnold R, et al.Autologous stem cell transplantation for systemic lupus erythemato-sus. Lupus 2004; 13: 168-176.

22. Burt RK, Traynor A, Statkute L, Barr WG, Rosa R, Schroeder J, et al.Nonmyeloablative hematopoietic stem cell transplantation for sys-

temic lupus erythematosus. JAMA 2006; 295: 527-535.23. Pavletic SZ, Illei GG. The role of immune ablation and stem cell

transplantation in severe SLE. Best Pract Res Clin Rheumatol 2005;19: 839-858.

24. Petri M, Jones RJ, Brodsky RA. High-dose cyclophosphamide with-out stem cell transplantation in systemic lupus erythematosus. Ar-thritis Rheum 2003; 48: 166-173.

25. Lisukov IA, Sizikova SA, Kulagin AD, Kruchkova IV, Gilevich AV,Konenkova LP, et al. High-dose immunosuppression with autolog-ous stem cell transplantation in severe refractory systemic lupuserythematosus. Lupus 2004; 13: 89-94.

26. Fassas A, Kimiskidis VK. Stem cell transplantation for multiple scle-rosis: what is the evidence? Blood Rev 2003; 17: 233-240.

27. Fassas A, Passweg JR, Anagnostopoulos A, Kazis A, Kozak T,Havrdova E, et al. Hematopoietic stem cell transplantation for mul-tiple sclerosis. A retrospective multicenter study. J Neurol 2002;249: 1088-1097.

28. Inglese M, Mancardi GL, Pagani E, Rocca MA, Murialdo A, SaccardiR, et al. Brain tissue loss occurs after suppression of enhancementin patients with multiple sclerosis treated with autologous haemato-poietic stem cell transplantation. J Neurol Neurosurg Psychiatry2004; 75: 643-644.

29. Fassas A, Anagnostopoulos A, Kazis A, Kapinas K, Sakellari I,Kimiskidis V, et al. Autologous stem cell transplantation in progres-sive multiple sclerosis - an interim analysis of efficacy. J ClinImmunol 2000; 20: 24-30.

30. Carreras E, Saiz A, Marin P, Martinez C, Rovira M, Villamor N, et al.CD34+ selected autologous peripheral blood stem cell transplanta-tion for multiple sclerosis: report of toxicity and treatment results atone year of follow-up in 15 patients. Haematologica 2003; 88: 306-314.

31. Nash RA, Bowen JD, McSweeney PA, Pavletic SZ, Maravilla KR,Park MS, et al. High-dose immunosuppressive therapy and autolog-ous peripheral blood stem cell transplantation for severe multiplesclerosis. Blood 2003; 102: 2364-2372.

32. Saccardi R, Mancardi GL, Solari A, Bosi A, Bruzzi P, Di BartolomeoP, et al. Autologous HSCT for severe progressive multiple sclerosisin a multicenter trial: impact on disease activity and quality of life.Blood 2005; 105: 2601-2607.

33. Blanco Y, Saiz A, Carreras E, Graus F. Changes of matrix metallo-proteinase-9 and its tissue inhibitor (TIMP-1) after autologous he-matopoietic stem cell transplantation in multiple sclerosis. J Neuro-immunol 2004; 153: 190-194.

34. Muraro PA, Douek DC. Renewing the T cell repertoire to arrestautoimmune aggression. Trends Immunol 2006; 27: 61-67.

35. van Laar JM, McSweeney PA. High-dose immunosuppressive thera-py and autologous progenitor cell transplantation for systemic scle-rosis. Best Pract Res Clin Haematol 2004; 17: 233-245.

36. Farge D, Passweg J, van Laar JM, Marjanovic Z, Besenthal C, FinkeJ, et al. Autologous stem cell transplantation in the treatment ofsystemic sclerosis: report from the EBMT/EULAR Registry. AnnRheum Dis 2004; 63: 974-981.

37. Tsukamoto H, Nagafuji K, Horiuchi T, Miyamoto T, Aoki K, TakaseK, et al. A phase I-II trial of autologous peripheral blood stem celltransplantation in the treatment of refractory autoimmune disease.Ann Rheum Dis 2006; 65: 508-514.

38. Komatsuda A, Kawabata Y, Horiuchi T, Motegi M, Ozawa M,Fujishima N, et al. Successful autologous peripheral blood stem celltransplantation using thiotepa in a patient with systemic sclerosisand cardiac involvement. Tohoku J Exp Med 2006; 209: 61-67.

39. Lim SH, Kell J, al-Sabah A, Bashi W, Bailey-Wood R. Peripheral

1597



www.bjournal.com.br

blood stem-cell transplantation for refractory autoimmune thrombo-cytopenic purpura. Lancet 1997; 349: 475.

40. Urban C, Lackner H, Sovinz P, Benesch M, Schwinger W, Dorn-busch HJ, et al. Successful unrelated cord blood transplantation in a7-year-old boy with Evans syndrome refractory to immunosuppres-sion and double autologous stem cell transplantation. Eur JHaematol 2006; 76: 526-530.

41. Hunh RD, Fogarty PF, Nakamura R, Read EJ, Leitman SF, Rick ME.High-dose cyclophosphamide with autologous lymphocyte-depletedperipheral blood stem cell (PBSC) support for treatment of refractorychronic autoimmune thrombocytopenia. Blood 2003; 101: 71-77.

42. Seeliger S, Baumann M, Mohr M, Jurgens H, Frosch M, Vormoor J.Autologous peripheral blood stem cell transplantation and anti-B-cell directed immunotherapy for refractory auto-immune haemolyticanaemia. Eur J Pediatr 2001; 160: 492-496.

43. Meivar-Levy I, Ferber S. Regenerative medicine: using liver to gen-erate pancreas for treating diabetes. Isr Med Assoc J 2006; 8: 430-434.

44. Murtaugh LC, Melton DA. Genes, signals, and lineages in pancreasdevelopment. Annu Rev Cell Dev Biol 2003; 19: 71-89.

45. Balamurugan AN, Bottino R, Giannoukakis N, Smetanka C. Pro-spective and challenges of islet transplantation for the therapy ofautoimmune diabetes. Pancreas 2006; 32: 231-243.

46. Otonkoski T, Gao R, Lundin K. Stem cells in the treatment ofdiabetes. Ann Med 2005; 37: 513-520.

47. Melton DA. Reversal of type 1 diabetes in mice. N Engl J Med 2006;355: 89-90.

48. Domenick MA, Ildstad ST. Impact of bone marrow transplantation ontype I diabetes. World J Surg 2001; 25: 474-480.

49. Couri CE, Foss MC, Voltarelli JC. Secondary prevention of type 1diabetes mellitus: stopping immune destruction and promoting beta-cell regeneration. Braz J Med Biol Res 2006; 39: 1271-1280.

50. Voltarelli JC, Couri CE, Stracieri AB, Oliveira MC, Moraes DA,Pieroni F, et al. Autologous nonmyeloablative hematopoietic stemcell transplantation in newly diagnosed type 1 diabetes mellitus.JAMA 2007; 297: 1568-1576.

51. Svendsen CN, Langston JW. Stem cells for Parkinson disease andALS: replacement or protection? Nat Med 2004; 10: 224-225.

52. Vastag B. Stem cells step closer to the clinic: paralysis partiallyreversed in rats with ALS-like disease. JAMA 2001; 285: 1691-1693.

53. Janson CG, Ramesh TM, During MJ, Leone P, Heywood J. Humanintrathecal transplantation of peripheral blood stem cells in amyo-trophic lateral sclerosis. J Hematother Stem Cell Res 2001; 10: 913-915.

54. Mazzini L, Fagioli F, Boccaletti R, Mareschi K, Oliveri G, Olivieri C,et al. Stem cell therapy in amyotrophic lateral sclerosis: a method-ological approach in humans. Amyotroph Lateral Scler Other MotorNeuron Disord 2003; 4: 158-161.

55. Mazzini L, Fagioli F, Boccaletti R. Stem-cell therapy in amyotrophiclateral sclerosis. Lancet 2004; 364: 1936-1937.

56. Smikodub AI. Experience of application of embryonic stem cells inALS. Cytotherapy 2004; 6: 431.

57. De Bellis R, Cordoba A, Bello L. Treatment of 12 patients with ALS,grafting cells through neuroendoscopy. Bone Marrow Transplant2006; 37 (Suppl 1): S133.

58. Appel SH, Engelhardt JI, Henkel JS, Luo Y, Brenner MK, Popat UR.Allogeneic hematopoietic stem cell transplantation. 2004. AmyotrophLateral Scler Other Motor Neuron Disor 2004; 5 (Suppl 2): 54.

59. Sargsyan SA, Monk PN, Shaw PJ. Microglia as potential contribu-tors to motor neuron injury in amyotrophic lateral sclerosis. Glia2005; 51: 241-253.

60. Voltarelli JC. Perspectivas de terapia cellular na esclerose lateralamiotrófica. Rev Bras Hematol Hemoter 2004; 26: 155-156.

Documents

The use of stem cells for the treatment of … SB, Braz J Med Biol Res...1579 Braz J Med Biol Res 40(12) 2007 Use of stem cells in autoimmune diseases Brazilian Journal of Medical