TeSR™-AOF 3D

StemSpan™-XF has been developed for the in vitro culture and expansion of human hematopoietic cells, when the appropriate growth factors and supplements are added. This allows users the flexibility to prepare medium that meets their requirements. StemSpan™-XF contains pre-tested human-derived and recombinant human (rh) proteins.

Using appropriate StemSpan™ Expansion Supplements, StemSpan™-XF may be used to expand CD34+ cells isolated from human cord blood, mobilized peripheral blood, or bone marrow samples, or to expand and differentiate lineage-committed progenitor cells to generate populations of erythroid, myeloid, or megakaryocyte progenitor cells.

Data Figures

Figure 1. Immunophenotyping of Cultured CD34⁺ Cells

Cord blood (CB)-derived CD34⁺ cells were purified using EasySep™ Human Cord Blood CD34 Positive Selection Kit II (Catalog #17896) and cultured in StemSpan™-XF (Catalog #100-0073) supplemented with StemSpan™ CD34⁺ Expansion Supplement (Catalog #02691) and UM171*. After 7 days, the cultured cells were stained with fluorescently labeled antibodies against CD34, CD90 and CD45RA, in addition to viability dye 7-AAD, and analyzed by flow cytometry. Sequential gates were used to determine the percentages of viable (A) CD34⁺ and CD34⁺CD90⁺CD45RA^- cells, based on fluorescence minus one (FMO) controls and (B) CD34^bright and CD34^brightCD90⁺CD45RA^- cells.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al. 2014.

Figure 2. StemSpan™ Media Support Greater Expansion of Human CD34⁺ and CD34^bright Cells Than Other Commercial Media

Purified CB-derived CD34⁺ cells were cultured for 7 days in StemSpan™ media (SFEM, SFEM II, AOF, and XF, orange bars), and in five media from other suppliers (Commercial Alternative, grey bars). All media were supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM171*. The (A) frequency and (B) cell expansion of viable CD34⁺ and CD34^bright cells in culture were analyzed by flow cytometry as described in Figure 1. Compared to the commercial alternatives tested, StemSpan™ media showed significantly higher expansion of CD34⁺ and CD34^bright cells (P < 0.05 when comparing StemSpan™ SFEM, SFEM II, and XF to five media from other suppliers, calculated using a one-way ANOVA followed by Dunnett’s post hoc test). The CD34bright cell population is enriched for functional stem/progenitor cells (See Figure 4). Data shown are mean ± SEM (n = 8).

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.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al. 2014.

Figure 3. StemSpan™ Media Support Equal or Greater Expansion of Primitive Human CD34^brightCD90⁺CD45RA^- Cells Than Other Commercial Media

Purified CB-derived CD34⁺ cells were cultured for 7 days in StemSpan™ media (SFEM, SFEM II, AOF, and XF, orange bars), and in five media from other suppliers (Commercial Alternative, grey bars). All media were supplemented with StemSpan™ CD34⁺ Expansion Supplement and UM171*. The (A) frequency and (B) cell expansion of viable CD34⁺CD90⁺CD45RA^- (solid) and CD34^brightCD90⁺CD45RA^- (dotted overlay) cells in culture were analyzed by flow cytometry as described in Figure 1. Compared to the commercial alternatives tested, StemSpan™ media showed similar or significantly higher expansion of CD34^brightCD90⁺CD45RA^- cells (P < 0.05 when comparing StemSpan™ SFEM II and XF to five media from other suppliers, calculated using one-way ANOVA followed by Dunnett’s post hoc test). The CD34^brightCD90⁺CD45RA^- cell population is highly enriched for functional stem/progenitor cells (See Figure 4). Data shown are mean ± SEM (n = 8).

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.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al. 2014.

Figure 4. Culture-Expanded CD34^bright Cells Have Higher Colony-Forming Potential Than Culture-Expanded CD34^med and CD34^- Cells

Purified CB-derived CD34⁺ cells (n = 2) were cultured in StemSpan™-XF with StemSpan™ CD34⁺ Expansion Supplement and UM171* for 7 days. Cultured cells were stained with an anti-CD34 antibody and 7-AAD for sorting. Anti-CD90, and CD45RA antibodies were also included, to be used in later analyses. Cells were either (A) bulk-sorted or (B,C) single cell index-sorted from CD34^bright, CD34^med, and CD34^- populations. Sorted cells were plated in colony-forming unit (CFU) assays using MethoCult™ H4034 Optimum (Catalog #04034). (A) Bulk-sorted cells were plated at 400 cells/mL in 6-well SmartDish™ plates (Catalog #27370) and (B,C) single cells were sorted directly into 96-well plates (one cell per well, prefilled with 100 µL of MethoCult™). After 14 days of incubation, colonies were counted with (A) STEMvision™ or (B,C) manually using an inverted microscope. (A) Shown are representative images of CFU assays from bulk-sorted cells of CD34^bright, CD34^med, and CD34^- populations. The frequency of colonies in each population from the single cell-sorted experiments are shown as (B) CFU frequency, calculated as the total number of colonies generated by the total number of individually sorted cells (shown respectively as a fraction above each bar), and (C) CFU output expressed as total numbers of CFU in each sorted population per original input CD34⁺ cell.

The culture-expanded CD34^bright cells showed (B) higher CFU frequency (~50%) than CD34^med cells (~20%) and CD34^- cells (~10%), and yielded more diverse and primitive colony types, including CFU-GEMM and BFU-E colonies, which were rarely generated from CD34^med and CD34^- cells. When phenotyping data, collected during sorting, was analyzed all CFU-GEMM and the majority of of BFU-E colonies were generated from the fraction enriched for LT-HSCs, the CD34^brightCD90⁺CD45RA^- cell population, while 33% of BFU-E colonies were generated from the fraction enriched for ST-HSCs, the CD34^brightCD90-CD45RA- cell population. Only small CFU-G and CFU-M colonies were generated from CD34^brightCD90^+/-CD45RA⁺ cells, which is consistent with publications suggesting this population contains only progenitor cells.

*Similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1μM. For more information including data comparing UM171 and UM729, see Fares et al. 2014.

Protocols and Documentation

Find supporting information and directions for use in the Product Information Sheet or explore additional protocols below.

Document Type

Product Name

Catalog #

Lot #

Language

Document Type

Product Information Sheet

Product Name

StemSpan™-XF

Catalog #

100-0073

Lot #

All

Language

English

Document Type

Safety Data Sheet

Product Name

StemSpan™-XF

Catalog #

100-0073

Lot #

All

Language

English

Applications

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.

Research Area

Workflow Stages

Workflow Stages for Hematopoietic Stem and Progenitor Cell Research

Cell Sourcing and Isolation

Expansion and Differentiation

Analysis