Figure 1. StemSpan™ B Cell Generation Protocol

Cord blood-derived CD34+ HSPCs are seeded on Day 0 in StemSpan™ SFEM II (Catalog #09605) containing StemSpan™ B Cell Differentiation Supplement 1 (Catalog #100-1251) and cultured for 14 days, followed by StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 2 (Catalog #100-1252) for an additional 14 days. Medium should be topped up after 3 - 4 days of culture followed by two half-medium changes every 3 - 4 days. On Day 28, cells are harvested and reseeded in StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 3 (Catalog #100-1253), followed by a top up with StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 4 (Catalog #100-1254) after 3 days for further differentiation to B cells.

Figure 2. B Lymphoid Progenitors Are Contained Within the Lineage Negative Cell Population After 14 Days of Culture

Cord blood-derived CD34+ cells (freshly isolated or from cryopreserved stock) were cultured in StemSpan™ SFEM II (Catalog # 09605) containing StemSpan™ B Cell Differentiation Supplement 1 (Catalog #100-1251) for 14 days. (A-D) Cells were harvested and analyzed by flow cytometry for the presence of lineage-negative cells (Lin-, to exclude non-B lineage cells) and the expression of B lymphoid progenitor markers including CD34, CD10, CD19, and CD24. A small population of early B cell progenitors (which may contain proB and preB cells) can be detected on gated Lin- cells. (E) The average frequency of Lin- cells on Day 14 was 36.6 ± 1.4% with a yield of 47.1 ± 3.2 Lin- cells per input CD34+ cell. The graph shows mean with standard error (n = 36).

Figure 3. Frequency and Yield of CD19+ B Cells After 28 Days of Culture

Day 14 B lymphoid progenitors were further differentiated into CD19+ B cells by culturing for an additional 14 days in StemSpan™ SFEM II (Catalog #09605) containing StemSpan™ B Cell Differentiation Supplement 2 (Catalog #100-1252). (A-D) Cells were harvested and analyzed by flow cytometry for expression of CD10, CD19, CD20, CD24, and IgM within the Lin- cell population to detect later-stage B cell progenitors, containing PreB cells and a small population of IgM+ immature B cells. (E) The average frequency of CD19+ cells on Day 28 was 21.3 ± 1.6% with a yield of 82.7 ± 11.5 CD19+ cells per input CD34+ cell. The graph shows mean with standard error (n = 36)

Figure 4. Frequency and Yield of CD19+IgM+ and Antibody-Secreting B Cells After 35 Days of Culture

On Day 28, CD19+ B cells were further differentiated into CD19+IgM+ and antibody-secreting cells (ASCs) by culturing for an additional 7 days in StemSpan™ SFEM II (Catalog #09605) containing StemSpan™ B Cell Differentiation Supplement 3 (Catalog #100-1253) followed by StemSpan™ SFEM II containing StemSpan™ B Cell Differentiation Supplement 4 (Catalog #100-1254). (A-D) On Day 35, cells were analyzed by flow cytometry for the expression of CD19, CD20, CD24, CD27, CD38, and IgM. (E) The frequency and yield of CD19+ (pan B cells), CD19+IgM+ (IgM+ B cells), and CD19+CD27+CD38++ B cells (memory-like B cells and ASCs) are shown. On average, the frequencies and yield per input CD34+ cells were 54.0 ± 2.7%;140.7 ± 19.1, 27.3% ± 1.3%; 65.3 ± 9.9, and 14.2% ± 1.3%; 36.5 ± 5.3, respectively. The graph shows means with standard error (n = 36).

Figure 5. Antibody-Secreting B Cells Generated After 35 Days of Culture

On Day 35, immunoglobulin-secreting cell frequencies of cultured B cells were determined by ELISpot assay. (A) Images of dual ELISpot assays (CTL ImmunoSpot®, IgM/IgG and IgA/IgE) for the detection of IgM (red) and IgG (blue) or IgA (red) and IgE (blue) antibody-secreting B cells (StemSpan™ SFEM II with StemSpan™ B Cell Differentiation Supplements 3 and 4, right) compared to negative control (StemSpan™ SFEM II without StemSpan™ B Cell Differentiation Supplements 3 and 4, left); 10,000 culture Day 35 cells per well were used. IgE ASCs were not detected. (B) The frequency and yield of ASCs are shown. On average 610 ± 67, 5 ± 1, and 317 ± 47 cells per 10,000 Day 35 cells secrete IgM, IgG, and IgA antibodies with a yield of 16.2 ± 2.4, 0.1 ± 0.04, and 5.2 ± 1.7 per input CD34+ cell, respectively. The graph shows mean with standard error (n = 8 - 33).