Low-dose insulin-like growth factor binding proteins 1 and 2 and angiopoietin-like protein 3 coordinately stimulate ex vivo expansion of human umbilical cord blood hematopoietic stem cells as assayed in NOD/SCID gamma null mice
© Fan et al.; licensee BioMed Central Ltd. 2014
Received: 31 October 2013
Accepted: 21 May 2014
Published: 30 May 2014
Insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) can enhance the ex vivo expansion of hematopoietic stem cells (HSCs) when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL). In order to determine the optimal dose and combination of IGFs, IGFBPs and ANGPTLs, serial dilution and full permutation of IGFBP1, IGFBP2, IGF2 and ANGPTL3 were applied on a cryopreserved umbilical cord blood mononuclear cell (UCB-MNC) ex vivo expansion system.
In this system, 4 × 105 cells/ml of UCB-MNCs were inoculated in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with standard basal cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO and supported by a bone marrow mesenchymal stromal cell layer.
Paradoxically, experiment results showed that the highest expansion of CD34+CD38−CD90+ primitive progenitor was stimulated by cytokine combination of SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3 at a low dose of 15 ng/ml IGFBP1 and 20 ng/ml IGFBP2 and ANGPTL3. This ex vivo expansion was further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ−/−) mice. Limiting dilution assay showed excellent correlation between the HSC ex vivo surface marker of CD34+CD38−CD90+ and the in vivo competitive repopulating unit (CRU) functional assay.
IGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate the expansion of CD34+CD38−CD90+ primitive progenitor at low dose. The optimal combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34+CD38−CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSCs due to consistency with the in vivo CRU functional assay.
Ex vivo expansion of umbilical cord blood (UCB) hematopoietic stem cells (HSCs) may overcome the obstacle of low cell dose for UCB transplantation in adults. Insulin-like growth factors (IGFs), insulin-like growth factor binding proteins (IGFBPs) and angiopoietin-like proteins (ANGPTLs) have been described previously to help enhance ex vivo expansion of HSCs when used with a standard cytokine cocktail of stem cell factor (SCF), thrombopoietin (TPO) and FLT3 ligand (FL) [1–10]. ANGPTLs and IGFBPs have also been demonstrated to enhance HSC in vivo migration and activity, supporting survival and replating capacity [11–14]. However, the optimal dose and combination of these novel cytokines have yet to be determined. Current doses of IGFBPs and ANGPTLs are in the range of 100 to 500 ng/ml. In terms of clinical application, these concentrations may not be optimal and would be costly. Hence, investigations into the optimal cytokine dose and combination of IGFs, IGFBPs and ANGPTLs are important.
In this study, serial dilution and full permutation were used to determine the optimal cytokine dose and combination for stimulation of ex vivo expansion of UCB-HSCs. This established cytokine dose and combination were then further validated in 8-week-old to 10-week-old nonobese diabetic/severe combined immunodeficiency interleukin 2 receptor gamma chain null (NOD/SCID-IL2Rγ−/−; NSG) mice.
Cryopreserved UCB mononuclear cells (4 × 105cells/ml) were suspended in serum-free Stemspan® medium (Stemcell technologies, vancouver, BC, Canada) supplied with a standard cytokine combination of 100 ng/ml SCF, 50 ng/ml FL and 100 ng/ml TPO (all three cytokines purchased from Peprotech, Rocky Hill, NJ, USA) and with individually varied doses and combinations of IGFBP1, IGFBP2, IGF2 and ANGPTL3 (these four cytokines purchased from R&D Systems, Minnneapolis, MN, USA). The cells were inoculated on a passage 3 to 5 BM-derived mesenchymal stromal cell layer and cultured in 37°C incubator for 12 days. The expanded cells were harvested at the end of 12 days and the adherent cord blood cells were detached after 1 minute of incubation at room temperature with 0.25% trypsin–ethylenediamine tetraacetic acid.
Flow cytometric analysis
All data were acquired using the cytomics FC500 flow cytometer (Beckman Coulter, Inc., Miami, FL, USA) and 10,000 events per sample were collected. Cell viability (AnnV/7AAD), hematopoietic primitive progenitors (CD34/CD38/CD90) of unexpanded and ex vivo expanded UCB and human cell multi-lineage reconstitution (CD45, CD34, CD71, CD15/66b, CD3 and CD19/20) in mice were analyzed using the same method mentioned in our previously published paper [15, 16].
Methylcellulose colony assays
NSG mice were purchased from Jackson Laboratories (Klaine, USA) by SingHealth Experimental Medicine Centre and were maintained in the same facility. All animal experiments were performed under the approval of the SingHealth Institutional Animal Care and Use Committee. The unexpanded and expanded UCB with four different cytokine combinations, ‘SCF + TPO + FL’, ‘SCF + TPO + FL + IGFBP2’, ‘SCF + TPO + FL + IGFBP2 + IGF2 + ANGPTL3’ and ‘SCF + TPO + F + IGFBP1 + IGF2 + ANGPTL3’ at doses of 20 ng/ml IGFBP2 and ANGPTL3, 15 ng/ml IGFBP1 and 10 ng/ml IGF2, were injected intravenously via the tail vein into sublethally irradiated (240 cGy) 8-week-old to 10-week-old NSG mice. Acidified water and cyclosporine A were administered to NSG mice orally and by intraperitoneal injection for prophylaxis of bacterial and fungal infection and graft versus host disease.
Harvesting of mouse bone marrow
At the end of the fourth month of transplantation, the mice were sacrificed using a carbon dioxide chamber. The femur and tibia were harvested and placed into cold RPMI medium (Invitrogen, Grand Island, NY, USA) immediately. Joint ends were cut and BM was flushed out with 10 ml of 2% fetal bovine serum–RPMI. Subsequently, contaminated red blood cells were lysed by ammonium chloride-based buffer before flow cytometric analysis.
The results are expressed as mean ± standard deviation. The significance between two groups was determined using the two-sample independent t test. P < 0.05 was defined as significant. In terms of multiple comparisons, Bonferroni’s test was used to correct the P value for the t test. The processing and statistical analysis of the data was performed using OriginPro 7.5 software (OriginPro, Inc., Northampton, MA, USA).
Low dose of IGFBP1, IGFBP2, IGF2 and ANGPTL3 is enough to boost the ex vivo expansion of CD34+CD38−CD90+ primitive progenitor cells
‘SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3’ is the optimal combination to enhance ex vivo expansion of CD34+CD38−CD90+ primitive progenitor cells
Equivalent human cell engraftment and multi-lineage reconstitution profile between unexpanded and expanded umbilical cord blood
Phenotypic marker of CD34+CD38−CD90+ can be used as a hematopoietic stem cell ex vivo detecting marker
In this study, IGFBP1, IGFBP2, IGF2 and ANGPTL3 have been demonstrated to stimulate ex vivo expansion of CD34+CD38−CD90+ primitive progenitor at a low dose of 15 ng/ml IGFBP1, 10 ng/ml IGF2 and 20 ng/ml IGFBP2 and ANGPTL3. The optimal cytokine combination comprises IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. In view of the excellent correlation between the HSC ex vivo surface marker of CD34+CD38−CD90+ and the in vivo CRU functional assay, the CD34+CD38−CD90+ phenotype can serve as an ex vivo surrogate surface marker for HSCs.
We showed that IGFBP1, IGFBP2, IGF2 and ANGPTL3 could stimulate the ex vivo expansion of HSCs or primitive progenitors rather than common progenitors and total cells, in agreement with recent reports [4, 6, 7, 9, 10]. However, this stimulation had a negative correlation to cytokine dose, where a high dose of cytokine induced low expansion of primitive progenitors. In fact, the optimal cytokine dose appeared at the lower range of 10 to 20 ng/ml. Compared with 100 to 500 ng/ml high-dose cytokine usage, this 10 to 20 ng/ml low dose will help to dramatically reduce the cost in clinical applications.
In addition, our data also showed that an expanded unit with the established optimal cytokine cocktail of ‘SCF + TPO + FL + IGFBP1 + IGFBP2 + ANGPTL3’ had the capacity of multi-lineage reconstitution except for T-lymphoid cells. Even though the statistical analysis showed that there was no significant difference on T-lymphoid cell differentiation between expanded units and unexpanded units, T-lymphoid reconstitution capacity from the unexpanded unit was better than from the expanded unit by direct observation. The explanation for this phenomenon could be due to the cytokine cocktail used in ex vivo expansion to drive cultured cells to expand and differentiate into the myeloid lineage rather than the T-lymphoid lineage (data not shown). This defect can be overcome by double cord blood transplantation with one expanded unit and one unexpanded unit as demonstrated in our previous publication [15, 16].
From the limiting dilution assay, we know that only about 70% of expanded CD34+CD38−CD90+ primitive progenitors will reconstitute in NSG mice. What then happens to that 30% of expanded CD34+CD38−CD90+ primitive progenitors? We postulate two possibilities; the first is that 30% of expanded CD34+CD38−CD90+ primitive progenitors lose their in vivo reconstitution capacity during ex vivo expansion even though they express the biomarker phenotypically, while the second possibility could be that 70% of expanded CD34+CD38−CD90+ primitive progenitors possess self-renewal, pluripotency and long-term reconstitution capacity, whereas only 30% of them have short-term hematopoietic reconstitution capacity.
IGFBP1, IGFBP2, IGF2 and ANGPTL3 can stimulate ex vivo expansion of CD34+CD38−CD90+ primitive progenitor at low dose with the optimal cytokine combination comprising IGFBP1, IGFBP2 and ANGPTL3 together with the standard cytokine cocktail of SCF, FL and TPO. The CD34+CD38−CD90+ phenotype can serve as a surrogate ex vivo surface marker for HSC detection due to consistency with the in vivo CRU functional assay.
competitive repopulating unit
hematopoietic stem cell
insulin-like growth factor binding protein
insulin-like growth factor
nonobese diabetic/severe combined immunodeficiency interleukin 2 gamma chain null (NOD/SCID-IL2Rγ−/−)
stem cell factor
umbilical cord blood.
The authors thank Bryan and Zheng Lin, staff of SingHealth Experimental Medicine Centre, for help with mice work. This work was supported by a Cancer Syndicate Grant (grant number SCS-T0049) and a Central Grant (NMRC/CG/SGH/2010).
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