Literature search
The procedure of study selection was presented in Fig. 1. A total of fifteen clinic trials were included at last. Twelve of them were designed to study the effects of stem cells expansion in vitro while the other three were focusing on the incubation of stem cells ex vivo.
Study characteristics
Fourteen phase I/II clinic trials [11, 14–26] and one phase II/III clinic trials [27] were included and their characteristics were presented in Table 1. One study [14] had a randomized control group while fourteen studies [11, 15–27] were single arm clinic trials for using historical controls. It’s worth mentioning that the historical controls were well matched for transplantation conditioning, disease status, age, graft size, HLA matching and performance score criteria. Children and adult patients were both included in eight studies [11, 15–17, 21, 24, 26, 27] while six studies [18–20, 22, 23, 25] only focused on adult patients. Twelve studies [11, 15, 17, 18, 20–27] used myeloablative conditioning whereas only one study [19] used nonmyeloablative regimens, and two [14, 16] used both of them. One manipulated UCB unit and one unmanipulated unit were co-infused to patients in eleven studies [14, 16–25]. Four studies [11, 15, 26, 27] used only one manipulated UCB unit. Only one study didn’t select stem cells with CD34 + or CD133 + biomarker before expansion in vitro [18].
Table 1
Characteristics of studies included in the meta-analysis.
Study | Phase | Patients (experimental) | Patients (control) | Age | Preconditioning | CBUs | Expanded cell type |
2002 Shpall | I | 37 | – | children/adult | MA | 1 | CD34+ |
2007 Lima | I/II | 24 | 24 | - | NMA/MA | 2 | CD133+ |
2008 Lima(BMT) | I/II | 10 | - | children/adult | MA | 1 | CD133+ |
2008 Lima(Blood) | I/II | 35 | 36 | children/adult | NMA/MA | 2 | CD133+ |
2010 Delaney | I | 10 | 20 | children/adult | MA | 2 | CD34+ |
2012 Lima | I | 31 | 80 | adult | MA | 2 | unselected |
2013 Delaney | I | 23 | 40 | children/adult | MA | 2 | CD34+ |
2014 Horwitz | I | 11 | 17 | adult | MA | 2 | CD133+ |
2016 Wagner | I/II | 17 | 111 | children/adult | MA | 2 | CD34+ |
2017 Anand | II | 19 | 86 | adult | MA | 1/2 | CD133+ |
2018 Stiff | II/III | 101 | 295 | children/adult | MA | 1 | CD133+ |
2018 Horwitz | I/II | 36 | 146 | children/adult | MA | 1 | CD133+ |
2013 Brunstein | I | 29 | - | adult | NMA | 2 | - |
2015 Popat | I | 22 | 31 | adult | MA | 2 | - |
2013 Cutler | I | 21 | 53 | adult | MA | 2 | - |
Procedures of stem cell manipulation
The manipulation procedures of UCB units have been summarized in Table 2. The effects of expansion or incubation determined the cell status such as cell number, cell viability and cell type. And the outcomes of UCBT were closely associated with the cell status. Most of the expansion studies added regular cytokines into their cell culture systems including stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand (Flt3L), [18]megakaryocyte growth and development factor (MGDF) were also used in early studies [11, 14]. However, the effects of regular cytokines cultured stem cells on speeding up time to neutrophil recovery were controversial [28–31]. Some new small molecules or feeder cell layers were added into the cell culture system to augment the effects of regular cytokines in later expansion clinic trials [18, 24].
Table 2
Details of manipulation methods.
Study | Cytokines | Additional intervention | Culture time |
2002 Shpall | SCF GCSF MGDF | - | 10 d |
2007 Lima | SCF GCSF TPO | - | 14 d |
2008 Lima(BMT) | SCF TPO Flt3L IL-6 | 5uM TEPA | 21 d |
2008 Lima(Blood) | SCF GCSF TPO | - | 14 d |
2010 Delaney | SCF TPO Flt3L IL-6 IL-3 | Delta1ext − IgG | 16 d |
2012 Lima | SCF TPO Flt3L IL-6 GCSF | MSC | 14 d |
2013 Delaney | nr | Delta1ext − IgG | nr |
2014 Horwitz | SCF TPO Flt3L IL-6 | 2.5 mM NAM | 21 d |
2015 Forcade | SCF FLT3L TPO GCSF | - | nr |
2016 Wagner | SCF TPO Flt3L IL-6 | SR-1 | 15 d |
2017 Anand | SCF TPO Flt3L IL-6 | 2.5 mM NAM | 21 d |
2018 Stiff | SCF TPO Flt3L IL-6 | 5uM TEPA | 21 d |
2018 Horwitz | SCF TPO Flt3L IL-6 | 2.5 mM NAM | 19–23 d |
2013 Brunstein | - | 1 µg/m C3aL | 15 min |
2015 Popat | - | 1 mM β-fucose | 30 min |
2013 Cutler | - | 10uM PEG2 | 60 min/120 min |
As for incubation studies, the cytokines mentioned above were not necessary. Three kinds of small molecules were used to improve the stem cells’ homing ability by incubating with UCB units from fifteen minutes to two hours. And the incubating time was vital. PGE2 could contribute to a better clinic outcome through two hours’ incubation with stem cells, however, the effects were not good as only one hours’ incubation. So the data of patients in one hours’ incubation group were not evaluated in our study.
Comparing the differences between two methods, the processes of UCB expansion often cost lots of materials including serum free mediums and cytokines. All these additive is known to be expensive and could increase the financial burden on patients at last. There is also high risk of contamination during the several days of cell culture. In fact, the contamination cases are often reported in expansion studies [22, 24]. As a result, the incubation procedures seemed to be cheaper, safer and more convenient in accelerating neutrophil recovery.
Described in Table 3, expansion procedures yielded an average of 1.7–854 fold expansion (FE) in TNCs and 2.2–330 FE in CD34 + cells. Manipulated UCB units had more TNC numbers than unmanipulated units. The difference was larger for CD34 + cells. As a comparison, incubating did not alter the cell numbers. Much more TNCs and CD34 + cells thus could be infused to patients after expansion manipulation. But can they lead to a better outcome in UCB transplantation?
Neutrophil recovery
The medium time and range of neutrophil recovery can be acquired from fifteen studies [11, 14–27] and the data of randomized or historical controls are available in ten studies. Although the evaluation including data from both treatment and control group is the gold standard for meta-analysis, more published bias will emerge with only these ten studies included. To make a more rounded analysis, we discreetly enrolled all fifteen studies including five studies that didn’t have control groups and regarding all of them as single-arm clinic trials. And the other outcomes will be evaluated in the same way.
The overall effect size (ES) of neutrophil recovery in expansion studies and incubation studies was 17.77 (95%CI 14.02–21.51) and 16.87 (10.04–23.70), respectively (Fig. 2A-B). No significant difference was found (P = 0.308). Expansion or incubation, compared with control group, could both accelerate the neutrophil recovery. But it seems the time to neutrophil recovery remained the same even these patients in expansion studies received much more TNCs and CD34 + cells.
Platelet recovery
The Overall ES was 47.83 (95%CI 38.80-56.86) for expansion manipulation group and 46.03 (95%CI 38.58–53.48) for incubation manipulation group. (Fig. 2C-D). No significant difference between two kinds of manipulation was found (P = 0.4754). The result revealed that the patient’s platelet recovery time in expansion groups was barely shortened after being infused so many expanded UCB stem cells comparing with the patients in incubation group.
100-day survival
Based on existing studies, eight expansion studies and two incubation studies reported the data of 100-days survival rate. The overall ES of 100-days survival rate was 0.91 (95%CI 0.84–0.97) for the expansion studies and 0.89 (95%CI 0.79–0.97) for the incubation studies. No significant difference between two manipulation procedures was found (P = 0.4216) (Fig. 3).
Relationship between cell dose and outcomes
The association between UCB cell dose and hematopoietic recovery was assessed by a list of studies [32–34]. The positive correlations between the two factors were reported, although not all of them were significant. This was further confined by a systematic review [35]. In our enrolled studies, Wagner et al, de Lima et al, and Horwitz et al reported the strong correlation between neutrophil recovery and infused TNC or CD34 + cell dose [18, 22, 24]. A moderate negative association between total infused TNC dose and neutrophil recovery in expansion studies was revealed (R2 = 0.4526 P < 0.05). The total TNC dose was unrelated to the neutrophil recovery in incubation studies. We also found a moderate correlation between CD34 + cell dose and neutrophil recovery in incubation studies (R2 = 0.9777 P = 0.095), however, not in expansion studies. Unlike the reported results in other reviews, no observable correlation between platelet recovery time and cell dose was revealed.
Mechanism involved in manipulation
The function and cell number of UCB units were believed to be altered by the small molecules and feeder cells. Not surprising, the involved intrinsic and extrinsic signals were different between two methods. And some common signals shared between expansion and incubation were also revealed (Fig. 4).
Delta-1ext − IgG, mesenchymal stem cells (MSC), SR-1, copper chelator, and nicotinamide (NAM) were used for stem cell expansion ex vivo. Hes-1,known as a Notch transcriptional target, could preserve the long-term reconstituting hematopoietic activity and the self-renewal ability of stem cells [36–40]. And the clinic trials could be able to be conducted with the application of immobilized Delta-1 ligand [41]. MSCs have been widely used for stem cell expansion as feeder layers [42–45]. As for the mechanisms that may be responsible for MSC’s regulating effects, no all agreed explanation was achieve. Y. K. Jang et al considered that a list of cytokines including IL-1α/β༌IL-6, IL-7, leukemia inhibitory factor (LIF), FL, TPO, SCF, C-kit, macrophage colony stimulating factor (M-CSF), and GM-CSF that secreted by MSCs could support the ex vivo expansion of stem cells [46]. High level of SDF-1α, an effective chemotactic factor which contributes to stem cell bone marrow homing, was also detected in the supernatants of UCB expansion system [47]. SR-1 could inhibit differentiation and promote proliferation of stem cells by antagonizing aryl hydrocarbon receptor (AhR) [48–50]. Copper chelator could reduce the intracellular copper content of stem cells by decreasing mitochondrial membrane potential, which lead to a better expansion result [51–54]. NAM could reduce the p21 expression and promote the proliferation of stem cells as one of the noncompetitive inhibitors of SIRT1. The homing ability was also increased by the regulation of CXCR4 downstream signaling pathways [55, 56]. Prostaglandin E2 (PGE2), C3a, and fucosylation were used for short time incubation to increase the bone marrow homing ability. PGE2 could upregulate the expression of Cox1 and Cox2, which contributes to stem cell proliferation [57]. The homing ability of stem cells was also increased by up-regulating CXCR4 after PGE2 incubation. Additionally, survivin, as the inhibitor of intracellular active caspase-3, was activated by PGE2 and thus suppressed the apoptosis of stem cells [58–60]. C3a-C3aR axis plays a functional role in enhancing marrow homing ability of stem cells by increasing their responsiveness to SDF-1. And the matrix metalloproteinase-9 (MMP-9) was involved in this process [61, 62]. Treatment of stem cells with the enzyme fucosyltransferase-VI and guanosine diphosphate fucose could increase the cell-surface sLeX determinants, further enhancing homing ability of stem cells through a better P- and E-selectin binding capacity [63–65].
Ongoing clinic trials
There are a total of seven clinic trials ongoing and all of them are using ex vivo expansion methods (NCT01590628, NCT01690520, NCT02730299, NCT03173937, NCT03441958, NCT04103879, NCT03913026). No clinic trial about incubation is available. Obviously, clinicians prefer expansion to incubation. Their belief might rely on the continuous development of cell culture techniques and application of effective new molecules, such as UM171 [79, 80]. Indeed, a trend toward better treatment outcomes along with the development of cell ex vivo culture system was emerged according to our cumulative meta-analysis (Fig. 5). But more clinic trials should be completed to prove the advantage of expansion methods.
Table 3
Expanded and infused cell dose.
Study
|
TNC EF medium and range
|
CD34+ EF medium and range
|
Infused manipulated TNC
|
Infused manipulated CD34+
|
Infused unmanipulated TNC
|
Infused unmanipulated CD34+
|
Infused total TNC
|
Infused total CD34+
|
2002 Shpall
|
56(1.03-278)
|
4.0(0.1-20.0)
|
nr
|
nr
|
nr
|
nr
|
0.99×107/Kg
|
1.04×105/Kg
|
2007 Lima
|
26(0.44-275)
|
2.2(0-18)
|
nr
|
nr
|
nr
|
nr
|
3.6×107/Kg
|
1.6×105/Kg
|
2008 Lima(BMT)
|
161(2-620)
|
2.26
|
nr
|
nr
|
nr
|
nr
|
1.8×107/Kg
|
1.5×105/Kg
|
2008 Lima(Blood)
|
23(0.44-275)
|
2.3(0-957)
|
nr
|
nr
|
nr
|
nr
|
3.5×107/Kg
|
1.8×105/Kg
|
2010 Delaney
|
645(192–2161)
|
140(36–688)
|
4.6×107/Kg
|
60.3×105/Kg
|
3.3×107/Kg
|
2.4×105/Kg
|
7.9×107/Kg
|
62.7×105/Kg
|
2012 Lima
|
12.2(1.0-29.8)
|
30.1(0-137.8)
|
5.84×107/Kg
|
9.5×105/Kg
|
2.5×107/Kg
|
8.6×105/Kg
|
8.34×107/Kg
|
18.1×105/Kg
|
2013 Delaney
|
nr
|
nr
|
nr
|
nr
|
nr
|
nr
|
nr
|
nr
|
2014 Horwitz
|
486(171-643)
|
72(16-186)
|
3.1×107/Kg
|
35×105/Kg
|
2.6×107/Kg
|
0.7×105/Kg
|
5.1×107/Kg
|
35.7×105/Kg
|
2015 Forcade
|
nr
|
nr
|
nr
|
nr
|
nr
|
nr
|
nr
|
nr
|
2016 Wagner
|
854(168-2121)
|
330(67-848)
|
5×107/Kg
|
175×105/Kg
|
2×107/Kg
|
4×105/Kg
|
7×107/Kg
|
182×105/Kg
|
2017 Anand
|
nr
|
nr
|
1.5×107/Kg
|
nr
|
2.5×107/Kg
|
nr
|
4×107/Kg
|
nr
|
2018 Stiff
|
401.5(0-764)
|
90(6-398)
|
nr
|
nr
|
nr
|
nr
|
3.06×107/Kg
|
1.41×105/Kg
|
2018 Horwitz
|
1.7
|
33
|
4.9×107/Kg
|
63×105/Kg
|
-
|
-
|
4.9×107/Kg
|
63×105/Kg
|
2013 Brunstein
|
-
|
-
|
1.5×107/Kg
|
3.2×105/Kg
|
2.5×107/Kg
|
3.4×105/Kg
|
4×107/Kg
|
7.6×105/Kg
|
2015 Popat
|
-
|
-
|
1.75×107/Kg
|
0.92×105/Kg
|
2.59×107/Kg
|
1.15×105/Kg
|
4.26×107/Kg
|
2.37×105/Kg
|
2013 Cutler
|
-
|
-
|
1.8×107/Kg
|
0.74×105/Kg
|
1.7×107/Kg
|
0.56×105/Kg
|
3.5×107/Kg
|
2.37×105/Kg
|