Efficient isolation of human CD34+CD38– hematopoietic stem cells using immunomagnetic sorting

Dariusz Krenz, Faidra Aivazidou, Andreas Bosio, Sebastian Knöbel, and Ute Bissels Miltenyi Biotec GmbH, Bergisch Gladbach, Germany

A B 100 Donor 1 Donor 1 Introduction Donor 2 Donor 2 80 80 Donor 3 Donor 3

1 60 60 cells

Hematopoietic stem and progenitor cells (HSPCs) stem cells and thus much more suitable for + cells

are an attractive source for cell-based therapies. functional analysis of stem cells, e.g., in expansion +

40 cells among 40

+ +

CD34 cells are the main source for functional protocols and engraftment assays. Also, the more % CD34

+ + – CD34 in vitro assays, although the majority of the CD34 primitive CD34 CD38 cells are being discussed as 20 20

cells are rather progenitor than stem cells. In contrast, an alternative cell source for –based % CD133 the CD34+ cell subpopulation with low CD38 therapy approaches with the potential to improve 0 0 CD34+CD38+ CD34+CD38– CD34+CD38+ CD34+CD38– expression (CD34+CD38– cells) is highly enriched for transduction protocols2,3.

Day 1 Day 7

10³ 10³ 1000 C 95.9% 32.5% 33.7% 10² 10² 750

+ + Results CD34 CD38 cells ioBlue ioBlue 500 V V 10¹ 10¹

250 CD45- CD45- 1 1 Side scatter 0 0 -1 -1 0 An easy two-step immunomagnetic protocol -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ + – 1 for isolation of CD34 CD38 cells CD34-FITC CD34-FITC CD133-PE 10³ 10³ 1000 98.8% 51.7% 75.3% + – CD34 CD38 cells were isolated from fresh human using the MACSQuant® Analyzer. Labeling Check 10² 10² 750 + + – cord by magnetic cell separation (MACS® Reagent is used for reliable detection of CD38 cells CD34 CD38 cells ioBlue ioBlue 500 V V Technology) using the CD34+CD38– Cell Isolation Kit. after their separation as binding of available CD38 10¹ 10¹ 250 CD45- CD45- CD34+CD38– cells were isolated in a two-step pro- antibody clones is sterically hindered by MicroBead 1 1 Side scatter 0 0 + tocol. First, CD34 cells were positively enriched labeling. The developed isolation protocol allows -1 -1 0 -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ using CD34 MultiSort MicroBeads. Subsequently, the separation of the two CD34+ cell subpopulations CD34-FITC CD34-FITC CD133-PE the MultiSort MicroBeads were enzymatically re- (fig. 1B). Starting from 1×108 mononuclear cord Figure 2 leased from the CD34 antibody. The isolated CD34+ blood cells (n = 7) with a CD34+ cell frequency cells were then labeled with CD38 MicroBeads to from 0.1% to 1%, we isolated 8.0×103 to 1.1×105 deplete CD38+ cells in a second separation step CD34+ cells with low CD38 expression and a mean The expansion rate of CD34+CD38– cells is higher (fig. 1A). Cells were fluorescently stained using CD34, CD34+ cell purity of 85% (range 63% to 94%). + + + compared to CD34 CD38 cells CD38, and CD45 antibodies, Labeling Check The number of CD34 cells with a high CD38 3 Reagent (LC), as well as propidium iodide to exclude expression ranged from 7.4×104 to 5.0×105 cells and + + – dead cells from analysis. Cell number and immu- a mean CD34+ cell purity of 82% (range 47% to 96%) The isolated CD34 cell subpopulations were also was higher with CD34 CD38 cells compared to + + nophenotype were analyzed by flow cytometry was measured. analyzed for their expansion rate during cultivation CD34 CD38 cells and the difference was more in 96-well plates. Expansion was calculated based on pronounced for CD34+CD133 + cells. Simultaneous A the number of cells seeded at day 1 and the number analysis of immunophenotype and cell divisions with 1st magnetic 1st magnetic Bead Stop 2nd magnetic 2nd magnetic of cells obtained after six (donor 3) or seven days carboxyfluorescein diacetate N-succinimidyl ester labeling separation release of release labeling separation (donor 1 and 2). Cell numbers were determined by (CFSE) will be performed in upcoming experiments. + CD34+ cells are Labeled cells are MultiSort The release CD34+ cells Target cells are flow cytometry after gating on viable CD34 cells (fig. This analysis will provide more insight into the magnetically enriched by MicroBeads are reaction is are then separated by 3A) or the more primitive CD34+CD133 + cells (fig. 3B). behavior of the primitive CD34+CD38– cells during labeled with using two LS released from stopped by magnetically using an LS The expansion rate of CD34+ and CD34+CD133 + cells in vitro cultivation. CD34 MultiSort Columns and a the cells by the adding the labeled with Column and a MicroBeads. MACS Separator. MultiSort MultiSort Stop CD38 MACS Separator. A B Release Reagent. MicroBeads. CD34+ cells CD34+CD133+ cells Reagent. Donor 1 Donor 1 Donor 2 Donor 2 The primary 40 40 antibody Donor 3 Donor 3 remains on 30 30 the cells. 20 20

10 10 Fold expansionFold expansionFold

0 0 CD34+CD38+ CD34+CD38– CD34+CD38+ CD34+CD38–

Figure 3

+ – + Colony-forming unit potential of CD34 CD38 B After CD34 cell enrichment, Before separation before CD38+ cell depletion CD34+CD38+ cells CD34+CD38– cells and CD34+CD38+ cells 10³ 10³ 10³ 10³ 4 0.4% 92.1% 94.2% 89.1% 10² 10² 10² 10² Isolated CD34+CD38– and CD34+CD38+ cells from four ioBlue ioBlue ioBlue ioBlue V V V V 10¹ 10¹ 10¹ 10¹ 40 + Donor A different donors were seeded in methylcellulose CD45- CD45- CD45- CD45- medium and cultured for 14 days (fig. 4). Subsequently, 1 1 1 1 30 Donor B 0 0 0 0 cultures were analyzed microscopically and the -1 -1 -1 -1 Donor C -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ 20 colonies were counted as well as classified. A slightly Donor D CD34-FITC CD34-FITC CD34-FITC CD34-FITC higher colony count was observed for the CD38+ 10³ 10³ 10³ 10³ cells seeded 10 subpopulation. The outcome for the different colony

10² 10² 10² 10² CD34 per 100 Colonies types that were classified as burst-forming unit 0

+ + + – erythrocyte (BFU-E), colony-forming unit granulo-

10¹ -APC 10¹ -APC 10¹ -APC 10¹ CD34 CD38 CD34 CD38

LC LC LC cyte (CFU-G), macrophage (CFU-M), or granulocyte- CD38-APC 1 1 1 1 macrophage (CFU-GM) was very similar for both 0 0 0 0 Figure 4 -1 -1 -1 -1 subpopulations analyzed. -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³ -1 0 1 10¹10² 10³

CD34-FITC CD34-FITC CD34-FITC CD34-FITC Figure 1

Isolated CD34+CD38– cells remain more primitive than Conclusion CD34+CD38+ cells during cultivation 2 • An easy two-step immunomagnetic isolation References protocol for CD34+CD38– cells was developed. 1. Reems, J.A. and Torok-Storb, B. (1995) Blood The isolated CD34+CD38– and CD34+CD38+ cells proportion of CD133+ cells within the CD34+ cell • Functional analysis of the isolated cells confirmed 85: 1480–1487. from three different donors were cultivated in population was up to 42% higher after cultivation the more primitive phenotype of the CD38– 2. Baldwin, K. et al. (2015) Stem Cells 33: 1532–1542. StemMACS™ HSC Expansion Media XF supplemented of CD34+CD38– cells (fig. 2B). Figure 2C shows subpopulation. 3. Zonari, E. et al. (2017) Stem Cell Reports with StemMACS HSC Expansion Cocktail for six a comparison of the CD34 and CD133 marker + – 8: 977–990. (donor 3) or seven days (donor 1 and 2). After expression on the cultivated CD34+CD38– and • CD34 CD38 cells maintain a more primitive + + cultivation, the frequency of CD34+ cells was up to CD34+CD38+ cells for donor 2. In summary, cultiva- immunophenotype than CD34 CD38 cells 28% higher when the culture was started with the ted CD34+CD38– cells maintain a more primitive during cell expansion. more primitive CD34+CD38– cells (fig. 2A). For CD133 immunophenotype than CD34+CD38+ cells. Unless otherwise specifically indicated, Miltenyi Biotec products and services are for research use only and not for therapeutic or diagnostic use. MACS, the MACS logo, StemMACS, and VioBlue are registered trademarks or trademarks of Miltenyi Biotec GmbH and/or its affiliates in various countries worldwide. Copyright © 2017 Miltenyi Biotec GmbH and/or its affiliates. expression this effect was even stronger as the All rights reserved.