Serum-free culture supplement for expansion of human megakaryocytes
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Formulated to produce large numbers of human megakaryocytes in liquid cultures initiated with CD34+ CB or BM cells.
Optimized for use with StemSpan™ media. When combined with StemSpan™ SFEM II in particular, supports up to 2-fold higher expansion of megakaryocytes from human CD34+ CB cells than other serum-free media on the market.
Supplied as a 100X concentrate. After thawing and mixing, the tube contents can be added directly to any hematopoietic cell expansion medium of choice.
StemSpan™ Megakaryocyte Expansion Supplement (100X) contains a combination of recombinant human cytokines formulated to selectively promote the expansion and differentiation of human megakaryocyte progenitor cells from CD34+ cells isolated from human cord blood (CB) or bone marrow (BM) samples.
StemSpan™ Megakaryocyte Expansion Supplement (100X) is intended for use in combination with any of the following StemSpan™ media:
• StemSpan™ SFEM (Catalog #09600)
• StemSpan™ SFEM II (Catalog #09605)
• StemSpan™-XF (Catalog #100-0073)
• StemSpan™-AOF (Catalog #100-0130)
When added to serum-free medium, StemSpan™ Megakaryocyte Expansion Supplement typically promotes the production of hundreds of megakaryocytes per input CD34+ cell in 14-day liquid cultures initiated with CD34+ human CB cells. See data tab for more details.
Contains • Recombinant human stem cell factor (SCF)
<br> • Recombinant human interleukin 6 (IL-6)
<br> • Recombinant human interleukin 9 (IL-9)
<br> • Recombinant human thrombopoietin (TPO)
Subtype Supplements
Cell Type Hematopoietic Stem and Progenitor Cells, Megakaryocytes
Area of Interest Stem Cell Biology, Transplantation Research
Formulation Category Serum-Free
Data Figures
Figure 1. Production of Megakaryocytes by Expansion and Lineage-Specific Differentiation of CD34+ Human Cord Blood Cells Cultured in StemSpan™ SFEM Containing Megakaryocyte Expansion Supplement
Flow cytometry dot plots showing expression of the hematopoietic stem and progenitor cell marker CD34 and megakaryocyte markers CD41a and CD42b (A) before and (B,C) after culture of CD34+ cord blood cells for 14 days in StemSpan™ SFEM containing Megakaryocyte Expansion Supplement. The frequency of CD34+ cells declined from 90% before culture to <3% after 14 days, in parallel with a gradual accumulation of CD41a+CD42b+ megakaryocytes from 80% before and after culture, respectively.
Table 1. Production of Megakaryocytes from CD34+ Human Cord Blood Cells Cultured in StemSpan™ SFEM Containing Megakaryocyte Expansion Supplement
Numbers and percent of CD41a+ cells produced after 14 days of culture of enriched CD34+ cells from 6 independent cord blood (CB) samples. *95% confidence limits, the range within which 95% of the results will typically fall.
Figure 2. Comparison of Megakaryocyte Expansion in Different StemSpan™ Media Containing Megakaryocyte Expansion Supplement
(A) Average numbers and (B) frequencies of CD41+ megakaryocytic cells normalized relative to the values obtained in StemSpan™ SFEM (grey bars) after culturing purified CD34+ cord blood cells (n=6) for 14 days in StemSpan™ SFEM, SFEM II (blue bars) and AOF (orange bars) media containing Megakaryocyte Expansion Supplement. Vertical lines indicate 95% confidence limits, the range within which 95% of results typically fall. *The numbers of CD41a+ cells were significantly higher in SFEM II (p<0.01, paired t-test, n=6) compared to SFEM and AOF medium.
Note: Data for StemSpan™-AOF shown were generated with the original phenol red-containing version StemSpan™-ACF (Catalog #09855). However internal testing showed that the performance of the new phenol red-free, cGMP-manufactured version, StemSpan™-AOF (Catalog #100-0130) was comparable.
This product is designed for use in the following research area(s) as part of the highlighted workflow stage(s). Explore these workflows to learn more about the other products we offer to support each research area.
Identification of unipotent megakaryocyte progenitors in human hematopoiesis. Miyawaki K et al. Blood 2017 MAR
Abstract
The developmental pathway for human megakaryocytes remains unclear and the definition of pure unipotent megakaryocyte progenitor is still controversial. Using single-cell transcriptome analysis, we have identified a cluster of cells within immature hematopoietic stem and progenitor cell populations that specifically express genes related to the megakaryocyte lineage. We used CD41 as a positive marker to identify these cells within the CD34(+)CD38(+)IL-3Rα(dim)CD45RA(-) common myeloid progenitor (CMP) population. These cells lacked erythroid and granulocyte/macrophage potential, but exhibited robust differentiation into the megakaryocyte lineage at a high frequency, both in vivo and in vitro The efficiency and expansion potential of these cells exceeded those of conventional bipotent megakaryocyte/erythrocyte progenitors. Accordingly, the CD41(+) CMP was defined as a unipotent megakaryocyte progenitor (MegP) that is likely to represent the major pathway for human megakaryopoiesis, independent of canonical megakaryocyte-erythroid lineage bifurcation. In the bone marrow of patients with essential thrombocythemia, the MegP population was significantly expanded in the context of a high burden of Janus kinase 2 mutations. Thus, the prospectively isolatable and functionally homogeneous human MegP will be useful for the elucidation of the mechanisms underlying normal and malignant human hematopoiesis.
miR-34a contributes to megakaryocytic differentiation of K562 cells independently of p53. Navarro F et al. Blood 2009 SEP
Abstract
The role of miRNAs in regulating megakaryocyte differentiation was examined using bipotent K562 human leukemia cells. miR-34a is strongly up-regulated during phorbol ester-induced megakaryocyte differentiation, but not during hemin-induced erythrocyte differentiation. Enforced expression of miR-34a in K562 cells inhibits cell proliferation, induces cell-cycle arrest in G(1) phase, and promotes megakaryocyte differentiation as measured by CD41 induction. miR-34a expression is also up-regulated during thrombopoietin-induced differentiation of CD34(+) hematopoietic precursors, and its enforced expression in these cells significantly increases the number of megakaryocyte colonies. miR-34a directly regulates expression of MYB, facilitating megakaryocyte differentiation, and of CDK4 and CDK6, to inhibit the G(1)/S transition. However, these miR-34a target genes are down-regulated rapidly after inducing megakaryocyte differentiation before miR-34a is induced. This suggests that miR-34a is not responsible for the initial down-regulation but may contribute to maintaining their suppression later on. Previous studies have implicated miR-34a as a tumor suppressor gene whose transcription is activated by p53. However, in p53-null K562 cells, phorbol esters induce miR-34a expression independently of p53 by activating an alternative phorbol ester-responsive promoter to produce a longer pri-miR-34a transcript.
In vitro expanded cells contributing to rapid severe combined immunodeficient repopulation activity are CD34+38-33+90+45RA-. Vanheusden K et al. Stem cells (Dayton, Ohio) 2007 JAN
Abstract
Expansion of hematopoietic stem cells could be used clinically to shorten the prolonged aplastic phase after umbilical cord blood (UCB) transplantation. In this report, we investigated rapid severe combined immunodeficient (SCID) repopulating activity (rSRA) 2 weeks after transplantation of CD34(+) UCB cells cultured with serum on MS5 stromal cells and in serum- and stroma-free cultures. Various subpopulations obtained after culture were studied for rSRA. CD34(+) expansion cultures resulted in vast expansion of CD45(+) and CD34(+) cells. Independent of the culture method, only the CD34(+)33(+)38(-) fraction of the cultured cells contained rSRA. Subsequently, we subfractionated the CD34(+)38(-) fraction using stem cell markers CD45RA and CD90. In vitro differentiation cultures showed CD34(+) expansion in both CD45RA(-) and CD90(+) cultures, whereas little increase in CD34(+) cells was observed in both CD45RA(+) and CD90(-) cultures. By four-color flow cytometry, we could demonstrate that CD34(+)38(-)45RA(-) and CD34(+)38(-)90(+) cell populations were largely overlapping. Both populations were able to reconstitute SCID/nonobese diabetic mice at 2 weeks, indicating that these cells contained rSRA activity. In contrast, CD34(+)38(-)45RA(+) or CD34(+)38(-)90(-) cells contributed only marginally to rSRA. Similar results were obtained when cells were injected intrafemorally, suggesting that the lack of reconstitution was not due to homing defects. In conclusion, we show that after in vitro expansion, rSRA is mediated by CD34(+)38(-)90(+)45RA(-) cells. All other cell fractions have limited reconstitutive potential, mainly because the cells have lost stem cell activity rather than because of homing defects. These findings can be used clinically to assess the rSRA of cultured stem cells.
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