Delayed hematopoietic recovery is the main factor precluding a wider use of cord blood (CB) transplants. We hypothesized that this delayed engraftment might not be related to an insufficient number of stem cells in the graft, but to an intrinsic difficulty of these cells to undergo differentiation. To test our hypothesis, 2 groups of children were compared; 12 received a CB transplant and 12 an adult bone marrow (BM) transplant. We studied neutrophil and platelet recovery and, at a median time of approximately 1 year after transplantation, the frequency of colony-forming cells (CFCs) and long-term culture initiating cells (LTC-ICs) in the BM of the 2 groups. Recipients of BM transplants received 1-log more cells and had significantly faster neutrophil and platelet recovery. Conversely, the frequency of committed and early progenitors was significantly higher in the BM of children given CB cells compared with BM transplant recipients (median count of CFC/2 × 104 BM mononuclear cells, 20 versus 11, P = .007; median count of LTC-IC/106 BM mononuclear cells, 8.2 versus 0.2 P = .001). CB, but not adult BM stem cells, can better restore the host hematopoietic progenitor cell reservoir; the delayed engraftment after CB transplantation may reflect the difficulty of CB progenitors to reprogram themselves toward differentiation.

Cord blood (CB) represents an alternative source of hematopoietic stem cells for allografts in patients affected by either malignant or nonmalignant disorders.1-8  Over the last decade, several biologic studies have also demonstrated that, with respect to their bone marrow (BM) counterparts, CB cells are enriched of in vivo long-term repopulating stem cells and, when compared in vitro with BM cells, they produce larger colonies, have higher recloning capacity, and have better capability of engraftment into the nonobese/severe combined immunodeficiency (NOD/SCID) mouse model.9-12  Despite these characteristics, patients undergoing CB transplantation (CBT) are exposed to a greater risk of graft failure and experience delayed neutrophil and platelet recovery when compared to those given BM allografts.1-8 

Several studies have documented that the success of CBT is strictly correlated with the number of cells infused; patients given the highest number of cells are those with the lowest risk of death from transplant-related complications.2-8  This finding has led to the attempt to expand CB stem cells. Intriguingly, experiments in NOD/SCID mice have shown that the frequency of human CD34+ cells found in the BM of the animal is higher after CBT than after a comparable dose of adult BM cells.9  We reasoned that this finding might not reflect an odd feature of the mouse model, deficient for supporting differentiated human hematopoiesis,13  but, rather, a specific characteristic of CB stem cells, more prone to expand themselves than to differentiate.

To test this hypothesis in the clinical setting, we analyzed the speed of engraftment and the hematopoietic reconstitution, measured as the frequency of colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs), in 12 children given CB transplants and in 12 receiving BM cells.

Patients

The study was approved by the Institutional Review Board. Two groups of patients were included in this study: group 1 consisted of children given CB transplants; group 2 included children who received BM cells. Patients' guardians gave informed written consent. Details of the characteristics of patients are reported in Table 1.

Table 1.

Clinical characteristics of patients




Group 1, CBT

Group 2, BMT

P
Patient age, mo (range)   63 (3-180)   88 (30-152)   NS  
Patient sex, M/F   6/6   7/5   NS  
Donor age, y (range)   0   25 (2-46)   —  
Donor sex (M/F)   9/3   7/5   NS  
Type of donor   7 RD/5 UD   6 RD/6 UD   NS  
Original disorders     
    Acute leukemia   6   6   NS  
    Chronic leukemia   0   2   
    Thalassemia major   5   4   
    SCID   1   0   
Conditioning regimen     
    TBI + chemotherapy   4   5   NS  
    Busulfan + chemotherapy   8   7   
No. of cells infused, × 107/kg (range)   3.7 (1.9-18)   50 (21-126)   .0001  
PMNs more than 0.5 × 109/L, d (range)   22.5 (16-39)   13 (9-23)   .0002  
PLTs more than 50 × 109/L, d (range)   47.5 (30-100)   27 (21-58)   .0007  
Grade II-IV acute GVHD   3 of 12   6 of 12   NS  
Limited chronic GVHD   0 of 12   2 of 12   NS  
HCMV infection
 
2 of 12
 
4 of 12
 
NS
 



Group 1, CBT

Group 2, BMT

P
Patient age, mo (range)   63 (3-180)   88 (30-152)   NS  
Patient sex, M/F   6/6   7/5   NS  
Donor age, y (range)   0   25 (2-46)   —  
Donor sex (M/F)   9/3   7/5   NS  
Type of donor   7 RD/5 UD   6 RD/6 UD   NS  
Original disorders     
    Acute leukemia   6   6   NS  
    Chronic leukemia   0   2   
    Thalassemia major   5   4   
    SCID   1   0   
Conditioning regimen     
    TBI + chemotherapy   4   5   NS  
    Busulfan + chemotherapy   8   7   
No. of cells infused, × 107/kg (range)   3.7 (1.9-18)   50 (21-126)   .0001  
PMNs more than 0.5 × 109/L, d (range)   22.5 (16-39)   13 (9-23)   .0002  
PLTs more than 50 × 109/L, d (range)   47.5 (30-100)   27 (21-58)   .0007  
Grade II-IV acute GVHD   3 of 12   6 of 12   NS  
Limited chronic GVHD   0 of 12   2 of 12   NS  
HCMV infection
 
2 of 12
 
4 of 12
 
NS
 

SCID indicates severe combined immunodeficiency; TBI, total body irradiation; PMN, polymorphonuclear granulocytes; PLT platelets; HCMV, human cytomegalovirus; NS, not significant; —, not applicable; RD, related donor; and UD, unrelated donor

We have also studied the hematopoietic reconstitution at 1 year and 5 years after transplantation in adults (age range, 20-45 years) receiving BM from either a related or an unrelated donor.

In children receiving CB cells from a family donor, graft-versus-host disease (GVHD) prophylaxis consisted of cyclosporine (CsA) alone, whereas in children given an unrelated donor CB transplant, the combination of CsA and steroids was used.

In children undergoing BM transplantation (BMT) and belonging to group 2, GVHD prophylaxis consisted of CsA alone, when the donor was a compatible sibling, and of the combination of CsA and short-term methotrexate, when the donor was an unrelated volunteer. All patients were given a myeloablative preparative regimen. Recombinant human granulocyte colony-stimulating factor was used in all cases, starting in the first week after transplantation.

At time of analysis of hematopoietic reconstitution, all patients were in remission of the original disorder and had 100% donor chimerism.

Functional assays

Standard methylcellulose-based semisolid assays (Methocult; Stem Cell Technologies, Vancouver, BC, Canada) were used to evaluate CFC progenitors. After 14 days of incubation at 37°C in a humidified 5% CO2 atmosphere, colonies were scored using an inverted microscope applying standard criteria for their identification.14 

LTC-IC assays were performed by seeding an aliquot of light-density cells in dishes over irradiated (1500 cGy) murine stromal cell lines (M210-B4 kindly provided by Dr C. Eaves, BC Cancer Research Center, Vancouver, BC, Canada) as previously described.15  LTC-ICs were maintained 3 days at 37° C, then switched to 33°C and fed weekly by replacement of one half of the growth medium (Myelocult; Stem Cell Technologies, with the addition of 10-6 M hydrocortisone) containing one half of the nonadherent cells, with fresh growth medium. After 5 weeks, adherent cells were trypsinized and combined with the nonadherent fraction. These harvested cells were washed and aliquots were assayed for frequency of clonogenic precursors, as described. This value provides a relative measurement of the number of LTC-ICs present in the original sample.15 

Statistical analysis

Statistical analysis was carried out using a Mann-Whitney 2-sample (nonmatched) test (NCSS package, J. L. Hintze, Kaysville, UT).

The main characteristics of the patients together with posttransplantation events are reported in Table 1. CB and BM recipients were studied at a median of 12 months (range, 6-17 months) and 10.5 months (range, 5-17 months) after transplantation, respectively (P = NS). The 2 groups showed comparable characteristics except for the significantly lower (P = .001) number of cells infused per kilogram recipient body weight in CB recipients. The median time to reach neutrophil and platelet recovery, shown in detail in Tables 2 and 3, was significantly longer in CB recipients (P = .0002 and P = .0007, respectively).

Table 2.

Detailed clinical characteristics and hematopoietic reconstitution following CBT


Patient no.

Disease

Patient age, mo

Cells infused, × 107/kg

Time to PMN recovery, d

Time to PLT recovery, d

Time from CBT, mo

CFCs, per 2 × 104

LTC-ICs, per 106
1   ALL   48   3   26   42   7   24   8  
2   ALL   52   3.8   24   48   16   20   9.4  
3   Thal   73   4.8   39   75   7   56   15.4  
4   SCID   3   18   21   54   9   15   3.4  
5   ALL   61   4.5   28   100   17   20   5.4  
6   Thal   180   1.9   21   44   12   39   10  
7   Thal   96   3.6   20   30   13   7   7.2  
8   ALL   93   2.7   24   47   12   20   11.6  
9   AML   9   9   17   55   8   10   8.4  
10   Thal   71   5.7   26   46   13   13   7.2  
11   Thal   40   6   18   42   6   45   5.4  
12   ALL   66   2.7   16   68   16   50   12.6  
Median    63   3.7*  22.5  47.5  12§  20  8.2 
Range
 

 
3-180
 
1.9-18
 
16-39
 
30-100
 
6-17
 
7-56
 
3.4-15.4
 

Patient no.

Disease

Patient age, mo

Cells infused, × 107/kg

Time to PMN recovery, d

Time to PLT recovery, d

Time from CBT, mo

CFCs, per 2 × 104

LTC-ICs, per 106
1   ALL   48   3   26   42   7   24   8  
2   ALL   52   3.8   24   48   16   20   9.4  
3   Thal   73   4.8   39   75   7   56   15.4  
4   SCID   3   18   21   54   9   15   3.4  
5   ALL   61   4.5   28   100   17   20   5.4  
6   Thal   180   1.9   21   44   12   39   10  
7   Thal   96   3.6   20   30   13   7   7.2  
8   ALL   93   2.7   24   47   12   20   11.6  
9   AML   9   9   17   55   8   10   8.4  
10   Thal   71   5.7   26   46   13   13   7.2  
11   Thal   40   6   18   42   6   45   5.4  
12   ALL   66   2.7   16   68   16   50   12.6  
Median    63   3.7*  22.5  47.5  12§  20  8.2 
Range
 

 
3-180
 
1.9-18
 
16-39
 
30-100
 
6-17
 
7-56
 
3.4-15.4
 

ALL indicates acute lymphoblastic leukemia; Thal, thalassemia; and AML, acute myelogenous leukemia. Other abbreviations are explained in the footnote to Table 1. 

*

P = .0001

P = .0002

P = .0007

§

P = NS

P = .007

P = .001

Table 3.

Detailed clinical characteristics and hematopoietic reconstitution following BMT


Patient no.

Disease

Patient age, mo

Donor age, y

Cells infused, × 108/kg

Time to PMN recovery, d

Time to PLT recovery, d

Time from BMT, mo

CFCs, per 2 × 104

LTC-ICs, per 106
13   AML   30   14   6.0   9   26   8   11   0  
14   Thal   96   3   4.0   10   28   5   25   3.7  
15   ALL   120   33   4.7   18   36   9   5   0.4  
16   CML   152   25   2.9   12   24   17   3   0  
17   Thal   72   46   12.6   13   24   6   13   1.2  
18   CML   80   46   7.5   14   35   13   11   0  
19   AML   40   28   12.0   23   58   7   6   0  
20   AML   108   45   4.5   12   21   11   7   0.5  
21   ALL   110   52   6.2   15   38   12   18   1  
22   Thal   133   6   2.1   14   32   11   11   0  
23   ALL   45   11   3.4   9   21   13   15   2  
24   Thal   66   8   5.4   13   26   10   9   0  
Median    88   19   5*  13  27  10.5§  11  0.2 
Range
 

 
30-152
 
3-52
 
2.1-12.6
 
9-23
 
21-58
 
5-17
 
3-25
 
0-3.7
 

Patient no.

Disease

Patient age, mo

Donor age, y

Cells infused, × 108/kg

Time to PMN recovery, d

Time to PLT recovery, d

Time from BMT, mo

CFCs, per 2 × 104

LTC-ICs, per 106
13   AML   30   14   6.0   9   26   8   11   0  
14   Thal   96   3   4.0   10   28   5   25   3.7  
15   ALL   120   33   4.7   18   36   9   5   0.4  
16   CML   152   25   2.9   12   24   17   3   0  
17   Thal   72   46   12.6   13   24   6   13   1.2  
18   CML   80   46   7.5   14   35   13   11   0  
19   AML   40   28   12.0   23   58   7   6   0  
20   AML   108   45   4.5   12   21   11   7   0.5  
21   ALL   110   52   6.2   15   38   12   18   1  
22   Thal   133   6   2.1   14   32   11   11   0  
23   ALL   45   11   3.4   9   21   13   15   2  
24   Thal   66   8   5.4   13   26   10   9   0  
Median    88   19   5*  13  27  10.5§  11  0.2 
Range
 

 
30-152
 
3-52
 
2.1-12.6
 
9-23
 
21-58
 
5-17
 
3-25
 
0-3.7
 

CML indicates chronic myelogenous leukemia. Other abbreviations are explained in the footnote to Table 2 

*

P = .0001

P = .0002

P = .0007

§

P = NS

P = .007

P = .001

The frequency of committed (ie, CFC) and early (ie, LTC-IC) progenitors found in the BM after transplantation was significantly higher in children given CB cells compared with BM recipients (median count of CFC/2 × 104 BM mononuclear cells [MNCs], 20 versus 11, P = .007; median count of LTC-IC/106 BM MNCs, 8.2 versus 0.2, P = .001). Values of CFCs and LTC-ICs for each patient included in the study are reported in Tables 2 and 3. As reference, in our laboratory the frequency of CFCs and LTC-ICs in the BM of healthy donors is 30 (range, 10-96)/2 × 104 MNCs and 34 (range, 10-84)/106 MNCs, respectively.

The frequency of LTC-ICs observed in adult patients who had received BM transplants is reported in Table 4. These results show that, in these patients, reconstitution of early hematopoietic progenitors is very slow, without a significant improvement over time and is not influenced by the original disease (Table 4). Thus, the early hematopoietic reconstitution does not differ between children and adults receiving BM transplants (Tables 2,3,4 provide details).

Table 4.

Long-term hematopoietic reconstitution in adults undergoing BMT




LTC-ICs, per 106 MNC
Patient no.
Disease
1 y after BMT
5 y after BMT
1   CML   0   2.6  
2   CML   2   0  
3   CML   0   2  
4   CML   0   —  
5   AL   0   0  
6   AL   2   2  
7   AL   0   0  
8   AL   2.2   1  
9   AL   0   0  
10   AA   1.5   2  
11   AA   0   0  
12   AA   0   2.1  
13   AA   1   2  
Median    0   0.8  
Range
 

 
0-2.2
 
0-2.6
 



LTC-ICs, per 106 MNC
Patient no.
Disease
1 y after BMT
5 y after BMT
1   CML   0   2.6  
2   CML   2   0  
3   CML   0   2  
4   CML   0   —  
5   AL   0   0  
6   AL   2   2  
7   AL   0   0  
8   AL   2.2   1  
9   AL   0   0  
10   AA   1.5   2  
11   AA   0   0  
12   AA   0   2.1  
13   AA   1   2  
Median    0   0.8  
Range
 

 
0-2.2
 
0-2.6
 

CML indicates chronic myelogenous leukemia; AL, acute leukemia; AA, aplastic anemia; and —, not done

The major limitations to a wider use of CB transplants, especially for adults or, in general, for patients with a body weight exceeding 40 kg, are represented by the delayed hematopoietic recovery and the lower probability of donor engraftment, infection, and hemorrhage accounting for most of the nonrelapse deaths of CB transplant recipients.2-8  The most immediate interpretation proposed to explain the delayed hematologic recovery is that CB transplant recipients are given 1-log fewer nucleated cells and CD34+ cells. To accelerate the hematopoietic recovery and to increase the engraftment rate of CBT, several investigators have attempted to “expand” CB progenitor cells ex vivo, but these approaches, at present, have not led to a consistent clinical practice.16-18 

In contrast with the slow speed of engraftment in clinical transplants, CB cells show higher proliferative potential in some functional assays. In fact, a higher frequency of CD34+ human cells can be recovered from the BM of NOD/SCID mice injected with CB cells than with adult BM cells.9  However, in the clinical practice, “engraftment” is the ability of the injected stem cells to generate a mature progeny detected in the bloodstream, whereas, in the NOD/SCID mouse model, “engraftment” is the frequency of human cells detected in the marrow or spleen or both.

We considered that the capacity of CD34+ cells from CB to expand in vivo after transplantation in the xenograft model NOD/SCID could reveal a specific attitude of CB stem cells, which tend to privilege self-renewal at the expense of differentiation and maturation. If this hypothesis were correct, the number of progenitors recovered months after the allograft should be higher in the marrow of patients given CB transplants.

Our results document that the hematopoietic reconstitution after the allograft, as shown by the frequency of early and committed hematopoietic progenitors, is much better in children given transplants of CB than in both children and adults who received BM cells. In particular, already 1 year after transplantation, CB recipients show a frequency of LTC-ICs not different from the values found in some healthy individuals. This has never been observed in adult patients receiving BM transplants, in whom the frequency of LTC-ICs remains very low up to 5 years after transplantation (Table 4; Podestà etal14 ).

It remains to be clarified why CB cells, which are capable of reconstituting the host progenitor cell pool one order of magnitude larger than that obtained with BM cells, are slow in producing mature blood cells. It is unlikely that the number of LTC-ICs infused could determine the difference observed in the hematopoietic reconstitution, because the average frequency of LTC-IC per MNC in CB has never been found higher than in BM.19  In addition, we have measured the total number of these progenitors in a series of patients given either CB or BM transplants and have found that recipients of BM cells receive on average one log more LTC-ICs than CB transplant recipients (M.P., unpublished results, 2003).

In conclusion, our results suggest that the conventional concept by which the delayed engraftment observed in CBT is due to an insufficient number of stem cells could no longer be tenable. Rather, the delayed engraftment after CBT may reflect the difficulty of CB progenitors to reprogram themselves toward differentiation.

Prepublished online as Blood First Edition Paper, April 10, 2003; DOI 10.1182/blood-2003-03-0720.

Supported in part by grants from the European fifth framework program EUROCORD II QLRT1999-00380 and Associazione Italiana Ricerca sul Cancro (AIRC), Consiglio Nazionale delle Ricerche (CNR), Ministero dell'Università e della Ricerca Scientifica e Tecnologica (MURST), and Istituto di Ricovero e Cura a Carattere Scientifico (IRCCS) Policlinico S. Matteo (F.L.) and AIREO, Eurocord III (F.F.).

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

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