transplantation of a combination of cd133+ and cd34+ selected progenitor cells from alternative...

Transplantation of a combination of CD133+ and CD34+ selectedprogenitor cells from alternative donors

Transplantation of positive selected haematopoietic stem cells

has gained wide acceptance in the treatment of leukaemias and

several non-malignant diseases (Urbano-Ispizua et al, 2001;

Gryn et al, 2002; Gaipa et al, 2003; Lang et al, 2003). The

method is a fundamental prerequisite for haploidentical

transplantation from mismatched-related donors, as profound

depletion of T and B cells facilitates the prevention of graft

versus host disease (GvHD) and Epstein–Barr virus (EBV)

lymphoproliferative disease (LPD) (Aversa et al, 1998; Hand-

gretinger et al, 2001; Aversa et al, 2002; Ortin et al, 2002). The

separation procedures commonly rely on antibodies against

the CD34 antigen expressed on pluripotent haematopoietic

precursor cells.

However, recent studies have revealed the existence of

CD34) stem cell populations that also have a repopulating

capacity and are putative precursors of CD34+ cells (Bhatia

et al, 1998; Zanjani et al, 1998). CD133, an important antigen

in this context, is a five transmembrane domain glycoprotein

that is mainly co-expressed with CD34 (Yin et al, 1997) but

also found on CD34)/CD38)/Lin) precursors (Gallacher et al,

2000).

Human CD133+/CD34)/Lin) cells are capable of giving rise

to CD34+ cells in vitro and engrafting sublethally irradiated

non-obese diabetic severe combined immunodeficient (NOD/

SCID) mice (Gallacher et al, 2000). Moreover, several studies

indicated that CD133+/CD34+ cells have a higher clonogenic

capacity, both in vitro and in vivo, than CD133)/CD34+ cells

(de Wynter et al, 1998; Gordon et al, 2003). In megakaryo-

poiesis, it has been demonstrated that the CD133+ subset

contains primitive cells that are able to efficiently produce all

categories of megakaryocyte progenitors (Charrier et al, 2002).

Finally, after in vitro stimulation, CD133+ selected progenitors

Peter Lang,1 Peter Bader,1 Michael

Schumm,1 Tobias Feuchtinger,1 Hermann

Einsele,2 Monika Fuhrer,3 Christof

Weinstock,4 Rupert Handgretinger,5 Selim

Kuci,1 David Martin,1 Dietrich

Niethammer1 and Johann Greil1

1Children’s University Hospital, University of

Tuebingen, Tuebingen, Germany, 2Department of

Internal Medicine, University of Tuebingen,

Tuebingen, Germany, 3Children’s University

Hospital, University of Munich, Munich,

Germany, 4Department of Transfusion Medicine,

University of Tuebingen, Tuebingen, Germany,

and 5St Jude Children Research Hospital,

Memphis, Tennessee

� 2004 Blackwell Publishing Ltd, British Journal

of Haematology, 124, 72–79

Received 14 August 2003; accepted for

publication 29 September 2003

Correspondence: Dr Peter Lang MD,

Department of Pediatric Oncology University

Children’s Hospital Eberhard Karl’s University,

Tubingen Hoppe Seyler Straße 1 D 72076

Tubingen, Germany.

E-mail: [email protected]

Summary

Positive selected haematopoietic stem cells are increasingly used for allogeneic

transplantation with the CD34 antigen employed in most separation

techniques. However, the recently described pentaspan molecule CD133

appears to be a marker of more primitive haematopoietic progenitors. Here we

report our experience with a new CD133-based selection method in 10

paediatric patients with matched unrelated (n ¼ 2) or mismatched-related

donors (n ¼ 8). These patients received a combination of stem cells

(median ¼ 29Æ3 · 106/kg), selected with either anti-CD34 or anti-CD133

coated microbeads. The proportion of CD133+ selected cells was gradually

increased from patient to patient from 10% to 100%. Comparison of CD133+

and CD34+ separation procedures revealed similar purity and recovery of target

populations but a lower depletion of T cells by CD133+ selection (3Æ7 log vs.

4Æ1 log, P < 0Æ001). Both separation procedures produced >90% CD34+/

CD133+ double positive target cells. Engraftment occurred in all patients

(sustained primary, n ¼ 8; after reconditioning, n ¼ 2). No primary acute

graft versus host disease (GvHD) ‡ grade II or chronic GvHD was observed.

The patients showed a rapid platelet recovery (median time to independence

from substitution ¼ 13Æ5 d), whereas T cell regeneration was variable. Five

patients are alive with a median follow-up of 10 months. Our data demon-

strates the feasibility of CD133+ selection for transplantation from alternative

donors and encourages further trials with total CD133+ separated grafts.

Keywords: stem cell transplantation, alternative donors, CD34+ selection,

CD133+ selection, haploidentical.

research paper

72 ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 124, 72–79

have been shown to convert into CD34) precursors with high

multilineage engraftment capacity in vivo. In the mouse model,

these cells gave rise to cells with T, B and natural killer (NK)-

phenotype that, to date, has never been observed for CD34+

selected progenitors (Kuci et al, 2003).

These findings raise the possibility that CD133 may be the

more important antigen in terms of multilineage engraftment

and that convincing results of transplantations with purified

CD34+ progenitors may in part be due to the fact that CD133

is co-expressed on the vast majority of these cells. Thus, our

aim was to investigate the clinical feasibility and safety of

CD133-based selection and transplantation in a small number

of patients by gradually increasing the proportions of CD133

selected progenitors in standard CD34+ selected grafts from

unrelated and mismatched-related donors. Furthermore, the

efficacy of CD34+ and CD133+ selection procedures was cross-

evaluated.

Patients and methods

Patients

Nine paediatric patients and one adult received transplants

consisting of a combination of CD34+ and CD133+ selected

progenitors from matched unrelated or from mismatched-

related donors between July 2001 and January 2003. Informed

consent was obtained from the legal guardians or patients, as

appropriate.

The histocompatibility of each patient and donor was

determined by high-resolution molecular [human leucocyte

antigen (HLA)-A, -B and-DRB1] typing methods. Two

patients had matched unrelated donors and eight patients

had one to three loci mismatched, haploidentical parent

(n ¼ 7) or sibling (n ¼ 1) donors. Age ranged from 1Æ2 to

38 years (median ¼ 10 years) and body weight from 9Æ7 to

84 kg (median ¼ 30 kg). Six patients had acute lymphoblastic

leukaemia (ALL; T ALL ¼ 3, B precursor ALL ¼ 3), two had

juvenile myelomonocytic leukemia (JMML), one had Wiskott-

Aldrich syndrome (WAS) and one had severe aplastic anaemia

(SAA) (Table I).

Stem cell mobilization and purification of progenitor cellsby CD34+ or CD133+ selection

All donors agreed to donate peripheral-blood stem cells

(PBSC). Donor PBSC were mobilized by administration of

1 · 10 lg/kg of granulocyte colony-stimulating factor

(G-CSF) daily for 5 d and were harvested by 1–3 leukapheresis

Table I. Diagnoses, donor mismatch, conditioning regimens and graft characteristics.

Patient

ID no. Diagnosis

Donor/HLA

mismatch

Conditioning

regimen

Total number

of stem cells/kg

CD34+ selected

cells/kg

CD133+ selected

cells/kg

Portion of CD133+

added to the graft (%)

1 ALL, CR3 MMRD

A, B, DR

TBI/TT/VP-16

OKT3/ATG Merieux�32Æ5 29Æ9 2Æ6 8

2 WAS MMRD

A, B, DR

Bu/Flud/Cy


3 ALL, CR1 MMRD

A

TBI/Flud/VP-16

ATG Fresenius�8Æ2 6Æ6 1Æ6 19

4 ALL, CR1 MMRD

A, B, DR

TBI/Flud/VP-16


5 JMML MMRD

B, DR

Bu/Flud/Cy


6 SAA MUD TLI (7 Gy)/Flud/Cy


7 JMML MUD Bu/Cy/Mel


8 ALL, CR1 MMRD

A, B, DR

TBI/Flud/VP-16


9 ALL, CR2 MMRD

A, B, DR

TBI/Flud/VP-16


10 ALL, graft failure MMRD

A, B, DR

TLI (7 Gy)/Flud


Total number of infused stem cells and amount of CD34+ and CD133+ selected cells · 10E6 per kilogram of patient body weight. The portions of

CD133+ selected cells added to the graft are shown in ascending order.

CR1 (2), first (second) complete remission; MMRD, mismatched-related donor; MUD, matched unrelated donor; TBI, total body irradiation; TLI,

total lympoid irradiation; Bu, busulphan; HLA, human leucocyte antigen; cyclophosphamide (Cy): 60 mg/kg/d ·2; etoposide (VP-16): 60 mg/kg;

thiothepa (TT): 10 mg/kg; fludarabine (Flud): 40 mg/m2/d ·4; anti-thymocyte globulin (ATG) Merieux�: 10 mg/kg/d ·3; ATG Fresenius�: 20 mg/

kg/d ·3, melphalan (Mel): 140 mg/m2; OKT3: 5 mg/m2/d ·10.

Transplantation of CD133+ Selected Progenitor Cells

ª 2004 Blackwell Publishing Ltd, British Journal of Haematology, 124, 72–79 73

procedures. We sought to obtain at least 10 · 106 progenitors

per kilogram patient body weight (bw). Additional mobiliza-

tions were necessary in six mismatched-related donors.

Selection of progenitors with anti-CD34 or anti-CD133 coated

microbeads was carried out with the automated CLINIMACS

device (Miltenyi Biotec, Bergisch Gladbach, Germany1 ). In the

first patient, about 10% of the collected progenitor cells were

purified with anti-CD133 microbeads. The portion of CD133+

selected cells was successively increased from one patient to the

next and reached 100% in the last patient. Each graft was split

into two fractions of varying proportions, of which one was

processed with anti-CD34 beads and the other with anti-

CD133 beads. Thus, a minimum of two column runs was

needed per patient.

When the number of residual T-cells was found to be

>2Æ5 · 104/kg bw, a second depletion step was performed as

previously described (Lang et al, 2002). Briefly, the selected

progenitor cells were adjusted to 20 ml of separation buffer

and incubated with 0Æ5 ml anti-CD3 magnetic beads (Dynal,

Hamburg, Germany) for 20 min. The solution was then placed

in a weak magnetic field and unbound progenitor cells

removed.

Before and after separation, cell populations were stained

with anti-CD34, anti-CD133, anti-CD3, anti-CD19 and

anti-CD45 monoclonal antibodies (mAbs) and analysed by

fluorescence-activated cell sorting (FACS) on FACScalibur

instruments (Becton-Dickinson, Munich, Germany) according

to the International Society for Hematotherapy and Graft

Engineering guidelines (Leuner et al, 1998). Debris, dead cells,

cell aggregates and platelets were excluded by gating on

forward and side light scatter and subsequently on CD45+

propidium iodide negative cells. A minimum of 50 000 events

was used for assessment. Cell viability was consistently >95%.

Treatment protocol

The myeloablative conditioning regimens were based on

either total body irradiation (TBI, six fractions of 2 Gy each)

or intravenous busulphan (12Æ8 mg/kg for age >3 years and

16 mg/kg for age <3 years), with specific modifications

according to individual diagnosis and age (Table I). The

adult ALL patient (ID no. 10) had rejected unmanipulated

bone marrow and peripheral stem cells from a matched

unrelated donor after myeloablative conditioning with TBI/

fludarabine (Flud)/cyclophosphamide (Cy). However, suc-

cessful engraftment was achieved with CD133+ cells from her

haploidentical sister after reconditioning with total lymphoid

irradiation (TLI, 7 Gy), Flud (30 mg/m2/d ·4) and anti-

thymocyte globulin (ATG)2 Fresenius� (Bad Homburg,

Germany; 10 mg/kg/d · 3). G-CSF was routinely adminis-

tered only in the first three patients. The other patients

received G-CSF only in case of severe infections (which

occurred only once, in patient no. 10). One patient with

JMML received a T cell-replete graft (10 · 106 cells/kg) from

a matched unrelated donor, followed by two doses of

methotrexate (MTX) and a short course of cyclosporin A

(CsA). Prophylactic post-transplant immunosuppression was

not required in any of the other patients. Supportive care was

carried out as previously described (Lang et al, 2003).

Assessment of engraftment, immune reconstitution andplatelet recovery

The day of engraftment was defined as the first of three

consecutive days on which the absolute neutrophil count

(ANC) was >0Æ5 · 109/l. Reconstitution of CD3+, CD4+,

CD8+, CD19+, and CD56+ lymphocytes was monitored by

weekly FACS analysis until T cell recovery began and was

subsequently assessed every 3 months.

Platelet recovery was defined as independence from platelet

substitution for at least 14 d, with a platelet count routinely

used to trigger such a transfusion of £20 · 109/l. The date of

the last platelet transfusion was taken as the first day of

recovery. The Kaplan–Meier method was used to evaluate the

recovery probabilities of the CD133+/CD34+ selected group

and of a historical cohort of patients from our institution.

This cohort comprised paediatric patients who consecutively

underwent transplantation between January 1995 and June

2001 with solely CD34+ selected progenitors from matched

unrelated and mismatched-related donors and for whom

platelet transfusion data were available (n ¼ 76). Further-

more, an additional control group was created out of this

cohort: to exclude patients with increased requirement for

platelet transfusions, those with haemorrhagic cystitis, venous

occlusive disease (VOD), aspergillosis, septicaemia with

positive blood cultures, or GvHD ‡grade II were not

considered for analysis. To equalize the amount of trans-

planted stem cells in the CD133+/CD34+ selected group and

in the CD34+ selected group, only patients who received

>8 · 106 cells/kg were accepted in the CD34+ selected control

group. The mean numbers of progenitors were

20Æ5 ± 9Æ4 · 106 cells/kg (CD34+ selected group) and

28Æ2 ± 20Æ3 · 106 cells/kg (CD133+/CD34+ selected group;

difference not significant, P ¼ 0Æ29). Boosts given after

secondary graft failure were excluded. All patients of the

CD34+ selected group (n ¼ 32) received G-CSF and the

median age and body weight was 6Æ3 years (0Æ4–24 years) and

19 kg (4–64 kg) respectively. The diagnoses were acute

leukaemias (n ¼ 20), chronic myeloid leukaemia (CML,

n ¼ 3) and non-malignant diseases (n ¼ 9).

Statistical analysis

Probabilities of survival and platelet recovery were evaluated

with the method of Kaplan and Meier. Kaplan–Meier curves of

platelet recovery were compared by using the log-rank test.

The Wilcoxon rank sum test was employed for a cross-

evaluation of purity, recovery and T cell depletion between

both separation methods. Results are given in medians (range)

unless otherwise indicated.

P. Lang et al


Results

Comparison of CD133+ and CD34+ separation procedures

The purity and phenotype of target cells, contamination by

residual T cells and recoveries in both methods were

compared (Table II). A total of 20 CD133+ separations

and 18 CD34+ separations yielded median purities of 93Æ4%

(62Æ1–98Æ4) and 97Æ5% (38Æ0–99Æ2) respectively (P ¼ 0Æ06). A

median of 0Æ09% (0Æ05–0Æ38) contaminating T cells were

detectable in the CD133+ selected stem cells, whereas CD34+

selection resulted in significantly less residual T cells [0Æ06%

(<0Æ01–0Æ16), P ¼ 0Æ002]. T cells were thus depleted more

efficiently by CD34+ selection (4Æ1 log) than by CD133+

selection (3Æ7 log, P < 0Æ001). B cells were depleted less

effectively than T cells and both methods produced similar

results. No differences were observed between the recoveries

of target cells (80Æ6% for CD133+ selection and 77Æ3% for

CD34+ selection). Figure 1 shows representative FACS

analyses after selection with anti-CD133 coated beads and

after selection with anti-CD34 coated beads. The vast

majority of stem cells was CD133+/CD34+ double positive

for all separations (CD133+ selection: 93Æ09%; CD34+

selection: 92Æ22%, Table III). Significant and convincing

populations of CD133+/CD34) cells were not detectable.

Small CD133)/CD34+ subpopulations were enriched by

CD34+ selection (1Æ5%) but not by CD133+ selection

(0Æ21%). Thus, consideration should be given to the fact

that the total number of yielded CD34+ progenitor cells will

be slightly lower after CD133+ selection than after CD34+

selection.

Graft composition

The patients received a combination of CD34+ selected and

CD133+ selected stem cells. The proportion of CD133+ selected

cells was increased from one patient to the next from about

10% in the first to 100% in the last (Table I). A total of 29Æ3(8Æ2–78Æ7) · 106 progenitor cells per kg bw were infused. These

numbers include stem cell boosts, which were given in six

patients 21–130 d after transplantation in order to stabilize the

donor-derived granulopoiesis without using G-CSF. Boosting

was considered if leucocyte counts fell under 1Æ0 · 109 cells/l.

The median number of residual T cells was 17 000 cells/kg

(6000–29 500). To reach this goal even in small children, a

second depletion step was carried out in four of 20 CD133+

separations (20%) and in two of 18 CD34+ separations (10Æ5%).

Engraftment

Initial engraftment occurred in 10 of 10 patients. The median

time to an ANC > 0Æ5 · 109/l without G-CSF stimulation was

28 d (range 16–36 d; n ¼ 6 patients).

Four patients received G-CSF (5 lg/kg) and had an

ANC > 0Æ5 · 109/l at 11 d (range 9–18). Eight of 10 patients

had sustained engraftment after initial transplantation.

Two patients with SAA and JMML, who had received

multiple platelet transfusions for more than 1 year prior to

transplantation, experienced late graft failure (rejection).

However, both patients were successfully regrafted by recon-

ditioning with steroids/OKT 3, ATG and T cell add-backs

(SAA) or by reinfusion of stem cells with T cell add-back alone

(JMML). Thus, all patients were finally engrafted.

Table II. Comparison of separation procedures.CD133+ selected (n ¼ 20) CD34+ selected (n ¼ 18) P-value

Total cell count

Pre 5Æ5 (2Æ0–8Æ7) · 1010 5Æ7 (3Æ6–11Æ8) · 1010 P ¼ 0Æ45

Post 258Æ5 (77Æ8–474Æ2) · 106 289Æ8 (85Æ8–775Æ4) · 106 P ¼ 0Æ83

Target cells

Pre 0Æ64 (0Æ2–1Æ01)% CD133+ 0Æ52 (0Æ27–0Æ81)% CD34+ P ¼ 0Æ48

Post 93Æ4 (62Æ1–98Æ4)% CD133+ 97Æ5 (38Æ0–99Æ2)% CD34+ P ¼ 0Æ06

Recovery 80Æ6 (42Æ4–114Æ2)% CD133+ 77Æ3 (40Æ5–97Æ7)% CD34+ P ¼ 0Æ49

T cells

Pre 28Æ4 (12Æ1–41Æ3)% 32Æ4 (22Æ7–43Æ6)% P ¼ 0Æ44

Post 0Æ09 (0Æ05–0Æ38)% 0Æ06 (0Æ002–0Æ16)% P ¼ 0Æ002

Depletion 3Æ75 (3Æ22–4Æ1) log 4Æ1 (3Æ5–5Æ6) log P < 0Æ001

B cells

Pre nd nd

Post 2Æ1 (0Æ5–9Æ2)% 1Æ5 (0Æ9–7Æ8)% P ¼ 0Æ67

Depletion nd nd

Target cells (CD133+ or CD34+), as well as CD3+ T cells and CD19+ B cells, were determined by

FACS analysis pre- and postenrichment (without second depletion step). Medians, ranges and

P-values of the Wilcoxon rank sum test are shown.

nd, not done; FACS, fluorescence-activated cell sorting.



Graft versus host disease

Primary acute GvHD. Seven patients (70%) showed no

symptoms of primary acute GvHD. Three patients (30%)

experienced grade I GvHD. Primary acute GvHD grade II–IV

was absent.

GvHD after infusion of donor T cells. The patient with SAA

received 5 · 105 donor T cells/kg bw from his matched

unrelated donor because of an increasing mixed chimaerism in

order to prevent another graft rejection. After this donor

leucocyte infusion he returned to complete donor type but

unfortunately experienced grade III GvHD.

Chronic GvHD. Patients were considered evaluable for chronic

GvHD if they engrafted and survived for 100 d. None of the

eight evaluable patients had chronic GvHD.

Survival

Five of the 10 patients are still alive (as at August 2003), with a

median follow-up of 10 months (range 9–18 months, Fig 2).

Four patients are free of disease. One patient relapsed after

transplantation and is currently treated with a mild chemo-

therapy regimen. The causes of death were relapse (n ¼ 2;

JMML patients ID nos 5 and 7), infection (n ¼ 2; adenoviral

hepatitis, ID no. 2; systemic adenoviral and fungal infection,

ID no. 1), or organ toxicity (n ¼ 1; bronchiolitis obliterans

Fig 1. (A) Immunophenotyping of target cells

after separation (double staining with anti-

CD34/anti-CD133). The cells were separated

with either anti-CD133 coated beads (left) or

with anti-CD34 coated beads (right). (B) In

several patients, fluorescence intensities of

CD133 low positive cells and the negative frac-

tion (defined by isotype controls) overlapped

and so potential CD133+CD/34) cells could not

be differentiated from negative cells. Such pop-

ulations (lower right quadrant) were considered

as not evaluable.

Table III. Subpopulations of target cells after separation [median

(range)], as detected by double staining (anti-CD34/anti-CD133).

CD133+ selected CD34+ selected

CD34+/133+ 93Æ09 (62Æ01–98Æ39)% 92Æ22 (37Æ93–97Æ68)%

CD34+/133) 0Æ21 (0Æ04–1Æ27)% 1Æ5 (0Æ08–10Æ25)%

CD34)/133+ ne ne

ne, not evaluable.

Fig 2. Overall survival: Kaplan–Meier estimate of the probability of

overall survival of CD133+/CD34+ selected patients.

P. Lang et al


organizing pneumonia in the adult patient ID no. 10). EBV

LPD, veno-occlusive disease or haemorrhagic cystitis were not

observed.

Platelet recovery and comparison with a historicalcontrol group

The median time to platelet recovery of the CD133+/CD34+

selected group was 13Æ5 d (Fig 3). Patients with (n ¼ 4) and

without (n ¼ 6) G-CSF stimulation showed similar recoveries

(15 vs. 13Æ5 d). One patient died from adenoviral hepatitis on

day 25 post-transplantation, before he had been independent

from platelet transfusion for at least 14 d.

These results from our CD133+/CD34+ selected group were

compared with those from a cohort of patients treated at our

institution with CD34+ selected stem cells between 1995 and

June 2001. The median time to platelet recovery of the whole

cohort was 32 d. Furthermore, all patients who had an

increased requirement for platelet transfusions or received

grafts with less than 8 · 106 CD34+ selected progenitors/kg

were excluded, as described in ‘Patients and Methods. The

median time to platelet recovery of this control group was

30 d. Thus, a faster recovery was observed in the CD34+/

CD133+ group than in the whole cohort (P ¼ 0Æ0027) or in the

selected control group (P ¼ 0Æ047).

Immune reconstitution

Figure 4 shows the immune recovery of all haploidentical

patients with haploidentical donors. CD56+ NK cells recovered

quickly, and a clear NK cell peak was observed within 1 month

after transplantation. No B-cell deficiency occurred and T-cell

recovery was variable among the patients. A subgroup of

patients who had received the current standard regimen [TBI,

etoposide (VP16), Flud and ATG Fresenius�, but not OKT3,

thymoglobulin (3 Merieux�, Lyon, France) or G-CSF, n ¼ 4]

showed a remarkably fast recovery of CD3+ T cells (mean

numbers 30, 60 and 90 d post-transplant: 0Æ038, 0Æ196 and

0Æ338 · 109 cells/l respectively).

Discussion

Our experience has shown CD34+ selection to be a useful tool

in producing minimal GvHD in both closely matched

unrelated and mismatched-related donors without any post-

transplant immunosuppression (Handgretinger et al, 2001;

Lang et al, 2003). However, primary engraftment (which has

been c. 85%) and recovery of platelets and T cells (delayed in

some patients) may be further optimized. We have thus

investigated the feasibility and safety of CD133+-based selec-

tion and transplantation in a small number of patients by

adding increasing proportions of CD133+ selected progenitors

to a standard CD34+ selected graft.

After mobilization with G-CSF, a predominantly CD133+/

CD34+ double positive donor progenitor population was

observed and CD133+ selection as well as CD34+ selection

resulted in similar purities and recoveries of these target cells.

Although both methods produced a profound depletion of

T cells, CD133+ selection was less effective than CD34+

selection. To not exceed our very low T cell threshold of

2Æ5 · 104 cells/kg, even in small children receiving extremely

high stem cell doses from haploidentical donors, we performed

a further reduction of T cells in 20% of CD133+ separations.

We could thus maintain a very low incidence of primary

GvHD in our CD133+/CD34+ patients, in agreement with the

incidence in patients who received solely CD34+ selected grafts

in the last 7 years. Thus, the selected progenitors are unlikely

to induce GvHD themselves and graft manipulations on the

basis of anti-CD133 mAbs may also prevent GvHD, provided

that a critical threshold of T cells is not exceeded.

Sustained engraftment after initial transplantation was

observed in eight of 10 patients, which corresponds to that

Fig 3. Platelet recovery: Kaplan–Meier estimates of the probability of

platelet recovery of CD133+/CD34+ selected patients compared

with that of a historical control group (only CD34+ selected).

P-value ¼ 0Æ047.

Fig 4. Immune reconstitution: reconstitution of T cells, natural killer

(NK) cells and B cells of all patients with haploidentical donors.

Absolute cell counts are shown. Points represent the mean values at

each time point.



of our CD34+ patient group (84%). The two patients who

rejected their grafts were successfully reconditioned without

causing organ toxicity. It is important to note that another

patient who experienced graft failure after transplantation of

unmanipulated bone marrow from an unrelated donor was

successfully engrafted with CD133+ selected stem cells from

her haploidentical sister. In several patients, decreasing neu-

trophil counts were observed 60–90 d post-transplant. Stem

cell boosts were capable of stabilizing the donor-derived

granulopoiesis without inducing GvHD in this situation.

However, further clinical studies must be carried out to

determine whether the use of CD133+ selected progenitors will

improve the engraftment rate.

We have so far not observed any adverse side-effects

attributable to CD133+ selection.

T-cell recovery was variable among our patients, although a

favourable T cell recovery was observed in all haploidentical

patients who had received ATG Fresenius� but no G-CSF.

Thus, the impact of CD133+ selected cells is unclear and the

use of G-CSF as well as the type of ATG may act as variables

that influence regeneration. G-CSF, in particular, has been

reported to interfere with cytokine production and regener-

ation of lymphocyte subsets (Volpi et al, 2001). Further studies

must be carried out to address this issue.

Another interesting finding was that patients with additional

CD133+ selected stem cells had a rapid platelet recovery.

Moreover, these patients even showed a tendency for faster

recovery than our historical cohort of patients transplanted

with CD34+ selected grafts. The exclusion of patients with

increased requirement for platelet transfusions or with lower

CD34+ cell doses did not abolish this tendency. Although

in vitro data provide support for this observation (Charrier

et al, 2002), some aspects need careful consideration: first, the

number of patients in our study is small. Secondly, the use of

growth factors has been reported to impair platelet recovery

(Keever-Taylor et al, 2001). However, this effect is unclear and

remains a subject of controversial discussions (Gisselbrecht

et al, 1994; Bernstein et al, 1998). In this report, all patients

with CD34+ selected grafts but only four of 10 patients of our

CD133+ selected group received G-CSF. Thus, the influence of

G-CSF may be unlikely but cannot definitely be ruled out.

Thirdly, it has been shown that a high CD34+ content of the

graft is associated with faster recovery (Bernstein et al, 1998).

This observation is in line with our mega dose concept, with

the implication that high stem cell doses contribute to

haematopoietic recovery. To eliminate this factor, we tried to

adjust the median stem cell dose of the CD34+ patient group

to that of the CD133+/CD34+ group. Although the difference

between both groups was no longer statistically significant, the

CD133+/CD34+ patients still received a slightly higher stem cell

dose (28 vs. 20 · 106/kg). Therefore, we cannot rule out that

the number of progenitor cells may also be involved in the

faster recovery of CD133+ patients.

Immunophenotyping revealed no striking difference

between CD133+ selected and CD34+ selected progenitors, as

both populations consisted predominantly of CD133+/CD34+

double positive cells. Although rare populations of CD34+/

CD133) cells were seen in CD34+ selected grafts, we were not

able to detect convincing populations of CD133+/CD34) cells

in CD133+ selected progenitors. However, the existence of this

subset has previously been demonstrated: low percentages were

found in cord blood (Gallacher et al, 2000) and inconsistently

in peripheral blood after G-CSF mobilization (Gordon et al,

2003; Handgretinger et al, 2003). It has to be taken into

consideration that our standard cytometry may have been

insufficient to detect such rare populations. Furthermore, an

overlap of fluorescence intensities did not allow differentiating

potential low positive CD133+/CD34) cells from the negative

fraction in some patients.

Apart from this, an interesting factor may be derived from

experimental data suggesting that antibody coated microbeads

might, to variable extents, activate intracellular signaling

pathways influencing proliferation and differentiation of

processed progenitors. Tada et al (1999) have demonstrated

that cross-linking of the CD34 antigen on the cell surface

induces an increase in tyrosine phosphorylation followed by

cap formation and enhanced cytoadhesion. Similar CD133-

mediated effects have, to our knowledge, not yet been

reported. Thus, interactions between target cells and the

antibodies used for their selection may be considered as

effectors of outcome, even in phenotypically identical cells.

It has to be mentioned that CD34+/CD133) cells were lost

by CD133+ selection. However, experimental data suggest that

conversion of double positive cells into CD34+/CD133) cells

in vivo may compensate for this.

In summary, we have demonstrated the feasibility of using

stem cell progenitors selected with anti-CD133 coated micro-

beads from alternative donors. The preliminary clinical results

presented here provide a basis for further studies in order

to evaluate the efficacy of exclusively CD133+ selected grafts.

Acknowledgments

We thank Shangara Lal for critical reviewing of the manuscript

and Olga Bartuli, Christiane Braun, Gabi Hochwelker, and

Ulrike Krauter for excellent technical assistance.

This work was supported by grants from the Deutsche

Forschungsgemeinschaft (SFB 510) and from the Reinhold

Beitlich Stiftung, Tuebingen, Germany.

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transplantation of a combination of cd133+ and cd34+ selected progenitor cells from alternative...

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