StemSpan™
StemSpan™
Hematopoietic Cell Expansion Media and Supplements
Why Use StemSpan™ Media and Expansion Supplements?
- Components are carefully selected to minimize lot-to-lot variability and support optimal and consistent culture results.
- Media do not contain cytokines, allowing the flexibility to add StemSpan™ Expansion Supplements, cytokines and/or additives.
- StemSpan™ SFEM II, combined with the appropriate Expansion Supplement, supports greater expansion of CD34+ cells and differentiation of erythroid cells, granulocytes, monocytes, megakaryocytes, T and NK cells than other media tested.
- StemSpan™-ACF is the first commercially available animal component-free medium for culturing HSPCs.
Expansion Media
StemSpan™ SFEM
Formulation:
Species:
- Human
- Mouse
- Rat
- Non-human primate
Recommended for:
Components:
- BSA
- Insulin
- Tranferrin
- Supplements
- IMDM base
StemSpan™ SFEM II
Formulation:
Species:
Recommended for:
Components:
- BSA
- Insulin
- Tranferrin
- Supplements
- IMDM base
StemSpan™ H3000
Formulation:
Species:
Recommended for:
Components:
- Human-derived and recombinant human proteins
- IMDM base
StemSpan™-ACF
Formulation:
Species:
Recommended for:
Components:
- Recombinant and synthetic components
- IMDM base
Expansion Supplements for Human HSPCs
Differentiation of Erythroid, Myeloid and Megakaryocyte Lineage Cells
Application:
Differentiation of T and NK Lineage Cells
Application:
Data
Figure 1. StemSpan™ SFEM II Serum-Free Expansion Medium Containing CC100 Cytokine Cocktail Supports Greater Expansion of Human CD34+ Cells Than Other Media Tested
Expansion of CD34+ cells normalized relative to the values obtained in StemSpan™ SFEM medium (dark gray bar) after culturing purified CD34+ CB cells for 7 days in StemSpan™ SFEM, SFEM II (gold bar) and ACF (orange bar), and six media from other commercial suppliers (light gray bars; Competitor 1-6, which included, in random order, StemLine II (Sigma), HPGM (Lonza), HP01 (Macopharma), SCGM (Cellgenix), StemPro34 (Life Technologies) and X-Vivo-15 (Lonza)). All media were supplemented with StemSpan™ CC100 Cytokine Cocktail. Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in StemSpan™ SFEM II were significantly higher than in all other media, except the numbers of CB cells produced in StemSpan™-ACF (*p<0.05, paired t-test, n=6).
Figure 2. StemSpan™ SFEM II Serum-Free Expansion Medium Containing CD34+ Expansion Supplement Supports Greater Expansion of Human CD34+ Cells Than Other Media Tested
Expansion of CD34+ cells normalized relative to the values obtained in SFEM medium (dark gray bar) after culturing purified CD34+ CB cells for 7 days in StemSpan™ SFEM, SFEM II (gold bar) and ACF (orange bar), and six media from other suppliers (light gray bars; Competitor 1-6, which included, in random order, StemLine II (Sigma), HPGM (Lonza), HP01 (Macopharma), SCGM (Cellgenix), StemPro34 (Life Technologies) and X-Vivo-15 (Lonza)). All media were supplemented with the StemSpan™ CD34+ Expansion Supplement. Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in StemSpan™ SFEM II were significantly higher than in all other media (*p<0.01, paired t-test, n=6).
Figure 3. StemSpan™ SFEM II Serum-Free Expansion Medium Containing Erythroid Expansion Supplement Supports Greater Expansion of Erythroid Cells Than Other Media Tested
The numbers of erythroid cells, normalized relative to the values obtained in StemSpan™ SFEM medium (dark gray bar), obtained after culturing purified CD34+ CB cells for 14 days in StemSpan™ SFEM, SFEM II (gold bar) and ACF (orange bar), and six media from other commercial suppliers (light gray bars; Competitor 1-6, which included, in random order, StemLine II (Sigma), HPGM (Lonza), HP01 (Macopharma), SCGM (Cellgenix), StemPro34 (Life Technologies) and X-Vivo-15 (Lonza)). All media were supplemented with StemSpan™ Erythroid Expansion Supplement. Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in StemSpan™ SFEM II were significantly higher than in all other media (*p<0.05, paired t-test, n=6).
Figure 4. StemSpan™ SFEM II Serum-Free Expansion Medium Containing Megakaryocyte Expansion Supplement Supports Greater Expansion of Megakaryocytes Than Other Media Tested
The numbers of megakaryocytes, normalized relative to the values obtained in StemSpan™ SFEM medium (dark gray bar), obtained after culturing purified CD34+ CB cells for 14 days in StemSpan™ SFEM, SFEM II (gold bar) and ACF (orange bar), and six media from other commercial suppliers (light gray bars; Competitor 1-6, which included, in random order, StemLine II (Sigma), HPGM (Lonza), HP01 (Macopharma), SCGM (Cellgenix), StemPro34 (Life Technologies) and X-Vivo-15 (Lonza)). All media were supplemented with StemSpan™ Megakaryocyte Expansion Supplement (Catalog #02696). Vertical lines indicate 95% confidence limits, the range within which 95% of results fall. The numbers of cells produced in the StemSpan™ media were significantly higher than in the other media (*p<0.01 paired t-test, n=6).
Table 1. Production of Myeloid Cells from Human CB CD34+ Cells Cultured in SFEM II Containing Myeloid Expansion Supplement or Myeloid Expansion Supplement II
Shown are numbers of total nucleated cells (TNCs) produced per input human CB-derived CD34+ cell and percentages of cells positive for myeloid markers CD13, CD14 and CD15 after 14 days of culture in SFEM II containing Myeloid Expansion Supplement (n = 14) or Myeloid Expansion Supplement II (n = 16). *95% confidence limits (CL); the range within which 95% of results typically fall.
Figure 5. Frequency and Yield of CD4 ISP and CD4+CD8+ DP Cells After 42 Days of Culture
CB-derived CD34+ cells (freshly isolated or frozen) were cultured with the StemSpan™ T Cell Generation Kit (Catalog #09940) for 42 days and (A) analysed by flow cytometry for the expression of CD4, CD8, CD3 and TCRαβ. The (B) frequency and (C) yield of CD4 ISP, double-positive (CD4+CD8+) and CD3+TCRαβ+-expressing double-positive cells (CD4+CD8+CD3+TCRαβ+) are shown. On average, 38% of the total viable population were DP (CD4++), of which 35% co-expressed CD3 and TCRαβ. The yields of total DP cells and CD3+TCRαβ+ DP cells per input CD34+ cell were ~23,000 and ~9,000, respectively. Shown are means with 95% confidence intervals (n = 31).
Figure 6. Frequency and Yield of CD8 SP T Cells After 49 Days of Culture
DP cells were further matured into CD8 SP T cells by culturing for an additional 7 days in StemSpan™ SFEM II with T Cell Progenitor Maturation Supplement (Catalog #09930), IL-15 (Catalog #78031) and ImmunoCult™ CD3/CD28/CD2 T Cell Activator (Catalog #10970) on coated plates. On day 49, cells were (A) analyzed by flow cytometry for the expression of CD3, TCRαβ, CD4 and CD8. The (B) frequency and yield of CD3+TCRαβ+-expressing cells and their subsets are shown. On average, 54% of the CD3+TCRαβ+ cells were DP (CD4+CD8+) and 38% were CD8 SP (CD4-CD8+). The average yield of CD8 SP T cells per input CD34+ cell was ~6,000. CD3+TCRαβ+ CD4 SP (CD4+CD8-) T cells were detected at very low frequencies (data not shown). Shown are means with 95% confidence intervals (n = 12).
Figure 7. Frequency and Yield of CD56+ NK Cells After 28 Days of Culture
CB-derived CD34+ cells (freshly isolated or frozen) were cultured with the StemSpan™ NK Cell Generation Kit for 28 days. Cells were harvested and analyzed for (A, B) CD56 and (A) NKp46 expression by flow cytometry. Dead cells were excluded by light scatter profile and viability staining. (B) The average frequency of viable CD56+ NK cells on day 28 was 77%, with ~9,000 CD56+ cells produced per input CB-derived CD34+ cell. Shown are means with 95% confidence intervals (n = 45: 23 freshly isolated and 22 frozen CD34+ cell samples). BM-derived CD34+ cells were also differentiated into NK cells using the StemSpan™ NK Cell Generation Kit. The yield of NK cells from BM HSPCs is typically lower than with CB, averaging ~75 per input CD34+ cell (n = 3, data not shown).
Key Applications
Generation of Mature Blood Cells in Vitro
Leberbauer et al. (2005) Different steroids co-regulate long-term expansion versus terminal differentiation in primary human erythroid progenitors. Blood 105(1)
SFEM II
Huijskens et al. (2014) Technical advance: ascorbic acid induces development of double-positive T cells from human hematopoietic stem cells in the absence of stromal cells. J Leukoc Biol 96(6)
CC100
Kumkhaek et al. (2013) MASL1 induces erythroid differentiation in human erythropoietin-dependent CD34+ cells through the Raf/MEK/ERK pathway. Blood 121(16)
CC110
Gaikwad et al. (2007) In Vitro Expansion of Erythroid Progenitors from Polycythemia Vera Patients Leads to Decrease in JAK2V617F Allele. Exp Hema 35(4)
Ex Vivo Expansion of HSPCs for Rapid/Sustained Hematopoietic Recovery Post Transplantation
Delaney et al. (2010) Notch-mediated expansion of human cord blood progenitor cells capable of rapid myeloid reconstitution. Nat Med 16(2)
Cutler et al. (2013) Prostaglandin-modulated umbilical cord blood hematopoietic stem cell transplantation. Blood 122(17)
Generating Target Cells for Reprogramming to Make Induced Pluripotent Stem Cells
Ohmine et al. (2011) Induced pluripotent stem cells from GMP-grade hematopoietic progenitor cells and mononuclear myeloid cells. Stem Cell Res and Therapy 2(6)
Gene Transfer into HSPCs
Lechman et al. (2012) Attenuation of miR-126 Activity Expands HSC In Vivo without Exhaustion. Cell Stem Cell 11(6)
SFEM II
Buechele C et al. (2015) MLL leukemia induction by genome editing of human CD34+ hematopoietic cells. Blood. Epub ahead of print