Q&A: Efficient CD34⁺ Cell Genome Editing and HPSC Maintenance

CRISPR-Cas9 gene editing of human hematopoietic stem and progenitor cells (HSPCs) holds promise for treating disease, either through cellular therapy or other methodologies. A critical part of developing a treatment using genome-edited HSPCs involves implementing an optimized gene editing and cell culture methodology. In their on-demand webinar, “Optimizing CD34⁺ Cell Genome Editing for Efficiency and HSPC Maintenance”, STEMCELL experts Dr. Marta Walasek and Ms. Danielle Nguyen Truong provide an overview of HSPCs and CRISPR-Cas9 genome editing, and describe the work carried out at STEMCELL Technologies to define optimal culture conditions for an efficient genome editing workflow.

Continue reading to find answers from webinar speakers, Dr. Walasek and Ms. Nguyen Truong, along with Hematology Product Manager, Ms. Selena Hallahan, from STEMCELL to some of the questions that were posed following the live webinar in April 2022. They have also supplied references for further reading.

Meet the Speakers

Dr. Marta Walasek

Dr. Marta Walasek

Dr. Marta Walasek is a Senior Scientist in the Hematology department at STEMCELL. She has over 10 years experience in the field of hematopoiesis and stem cell biology. Marta’s work focuses on the development of products for hematopoietic stem and progenitor cell expansion, differentiation, and readout.

Ms. Danielle Nguyen Truong

Ms. Danielle Nguyen Truong

Danielle Truong is a Technical Scientist in the Business Operations department at STEMCELL. She has over 12 years experience in the field of molecular biology and genome editing. Danielle’s work focuses on protocol development of genome editing on a variety of cell types, including stem and primary cells, using ArciTect™ CRISPR-Cas9.

CD34⁺ cell sourcing and isolation

Can you speak to the difference between mobilized CD34⁺ cells and those from bone marrow if the desired end product is differentiated erythroid cells?

Dr. Marta Walasek: When appropriate culture conditions and expansion medium are used, differentiation of either mobilized peripheral blood (mPB) or bone marrow (BM) to the erythroid lineage results in a highly pure population of differentiated erythroid cells. These can be identified by expression of CD71 and GlyA cell surface markers and by expression of mainly adult-type (beta-) hemoglobin. The cell yield is donor dependent. Additionally, there may be differences in HSPCs isolated from BM and mPB as suggested in the paper below by Mende et al.

Can this culture system for CD34⁺ expansion be used for hPSC-derived CD34⁺ cells?

Dr. Marta Walasek: StemSpan™ CD34⁺ Expansion Supplement (Catalog #02691) has been developed and optimized to promote expansion of primary CD34⁺ cells derived from cord blood (CB) or BM. The supplement has not been tested for the expansion of human pluripotent stem cell (hPSC)-derived CD34⁺ cells, which may have different growth factor requirements than primary cells. CD34⁺ expansion may also depend on the method used to generate the CD34⁺ cells from hPSCs.

What is the usual CD34 purity using the EasySep™ system?

Dr. Marta Walasek: The purity of CD34⁺ cells isolated with the EasySep™ system depends on whether the cells were isolated using positive or negative selection methods. Additionally, the purity and number of obtained CD34⁺ cells is dependent on quality, age, and size of the start sample. On average, for 36 - 48 hours-old cord blood samples, EasySep™ positive selection results in 87% (±12%) pure CD34⁺ cell population when using EasySep™ Human Cord Blood CD34 Positive Selection Kit III (Catalog #17897) or 93.5% (±1.1%) when using EasySep™ Human CD34 Positive Selection Kit II (Catalog #17856). In contrast, the purity of CD34⁺ cells obtained via negative selection with EasySep™ Human Progenitor Cell Enrichment Kit II (Catalog #17936) is on average 78% (±16%).
Frequently Asked Questions on Primary Cells

Frequently Asked Questions on Primary Cells

Have questions about sourcing CD34⁺ cells? Find answers to frequently asked questions about ready-to-use fresh or cryopreserved primary cells from STEMCELL.

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CD34⁺ cell culture

How does the marker expression of CD34/CD90/CD45RA typically change over the first 4 - 7 days in culture?

Dr. Marta Walasek: HSPCs in non-cultured samples can be identified based on the CD34⁺CD38⁻CD45RA⁻CD90⁺CD49f⁺ phenotype, as shown by Fares and coworkers (see reference below). However, the cellular phenotype changes during cell culture, reflecting the progression through the differentiation process. For example, cells may acquire CD45RA and lose CD90 marker expression, and eventually lose CD34 expression. The kinetics of the changes in CD34, CD45RA, and CD90 marker expression will depend on culture conditions and the duration of culture. In general, shorter culture is recommended when maintenance of stem and progenitor cell phenotype and function is prioritized over cell yields. In other words, the frequency of stem cells may be higher with shorter culture, but at the cost of low cell numbers. Additionally, antigen expression on cultured cells may not be as predictive for determining non-differentiated status or lineage potential when compared to antigen expression on CD34⁺ cells that have not been cultured. For example, primary CD34⁺ cells with low or undetectable CD38 expression (CD34⁺CD38⁻ phenotype) are highly enriched for hematopoietic stem cells and primitive progenitor cells, but the CD34⁺CD38⁻ phenotype of cultured cells may not be as primitive. In recent years, several markers, such as endothelial protein C receptor (EPCR), have been shown to enrich HSPCs following cell culture.

Fares I et al. (2017) EPCR expression marks UM171-expanded CD34⁺ cord blood stem cells. Blood 129(25): 3344–51.

Why is UM171 not available anymore?

Selena Hallahan (Product Manager, Hematology): UM171 was discontinued for sale by STEMCELL in October 2019 per the wishes of the patent holder.

Regarding the culture protocol outlined in the webinar, similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 10X the concentration of UM171 (i.e. 1 μM). The equivalency of these molecules in expanding human cord blood cells has been shown by Fares and coworkers and confirmed by STEMCELL.

Please note that STEMCELL is the only authorized retailer of UM729. All other vendors are selling these products without the express permission of the patent holder.

Is UM171 used in place of StemSpan™ CD34⁺ Expansion Supplement or in addition to it?

Selena Hallahan: UM171 is the small molecule that was used in addition to StemSpan™ CD34⁺ Expansion Supplement (Catalog #02691).

Please note that UM171 is no longer available for sale by STEMCELL; however, similar results are expected when using UM729 (Catalog #72332) prepared to a final concentration of 1 μM.

Will the expansion media components interfere with HSPC differentiation?

Selena Hallahan: Culture conditions may impact the differentiation of HSPCs. The choice of growth factors and supplements added to the culture media affects whether the cell is stimulated to expand or differentiate. Early-acting cytokines and compounds that prevent differentiation can be used to expand HSPCs, whereas lineage-specific differentiation is achieved with the use of lineage-specific growth factors, such as erythropoietin (EPO) to stimulate erythroid differentiation.

StemSpan™ serum-free media have been developed to support the expansion and differentiation culture of hematopoietic cells and do not contain hematopoietic growth factors. This allows users the flexibility to supplement the medium with growth factors and/or other stimuli of choice to promote expansion or lineage-specific differentiation of HSPCs.

StemSpan™ expansion supplements are pre-mixed cocktails of recombinant human cytokines and other additives. Each has been formulated to selectively promote the expansion of CD34⁺ stem and progenitor cells, or to stimulate their differentiation into erythroid, myeloid (granulocyte or monocyte), or megakaryocyte progenitor cells, when added to StemSpan™ media.

Together, StemSpan™ media and supplements support greater expansion of CD34⁺ cells and differentiation of erythroid cells, granulocytes, monocytes, and megakaryocytes, than other media tested.

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What was the medium used when plating the pre-editing culture?

Dr. Marta Walasek: We tested various StemSpan™ media as well as alternative commercial media to identify optimal culture conditions for genome editing. While all StemSpan™ media and supplements were compatible with the genome editing workflow, we found that StemSpan™ SFEM II (Catalog #09605) supplemented with StemSpan™ CD34⁺ Expansion Supplement (Catalog #02691) and the small molecule, UM729 (Catalog #72332), for 2 days prior to CRISPR-Cas9 editing best supports maintenance of CD34 expression and expansion of primitive HSPC subsets during genome editing.

More information on the culture conditions tested can be found in this technical bulletin.

CRISPR-Cas9 gene editing of CD34⁺ cells

Have you ever coupled lentivirus infection of an sgRNA with electroporation of Cas9? If so, how do you recommend we do it?

Danielle Nguyen Truong: We have developed a protocol for genome editing using CRISPR-Cas9 with electroporation of the RNP system into CD34⁺ cells. We have not coupled lentivirus infection of an sgRNA with electroporation of Cas9. It has been shown that lentiviral transduction of Cas9 is less efficient in sensitive cell types such as HSPCs. More information about lentivirus infection of sgRNA with electroporation of Cas9 has been described by Yudovich and coworkers.

Different types of donor templates can induce significantly different levels of cytotoxicity. Do you have any suggestions about electroporation conditions during AAV delivery?

Danielle Nguyen Truong: Homology-directed repair (HDR) efficiency is usually higher with short single-stranded oligodeoxynucleotide (ssODN) donor templates, which are often used to correct a single base change, or short insertions or deletions. HDR is less efficient when double-stranded (dsDNA) templates are introduced, and a high concentration of dsDNA can cause cytotoxicity in cells. Shy et al. developed a novel system that takes advantage of lower toxicity with single-stranded DNA (ssDNA), in which the hybrid ssDNA HDR templates were incorporated with Cas9 target sequences (CTS) to improve the knock-in efficiency and reduce toxicity in cells.

What is the delivery efficiency of the lentiviral system in CD34⁺ cells? What is the best viral vector for CD34⁺ cell transduction?

Danielle Nguyen Truong: Lentiviral systems generally have high editing efficiency and are effective in a variety of cell lines, primary cells, and stem cells. Yudovich and coworkers used Cas9 pLentiCRISPRv2 (pLCv2) to deliver sgRNA, followed by the transient expression of Cas9 using electroporation; the knockout efficiency of CD45 and CD44 was about 90% in edited HSPCs. However, the biggest concern of the lentivirus system is the random integration into the genome, which may disrupt vital genes or alter the regulation of gene expression. This suggests that non-integrating lentiviral vectors should be used for delivery of CRISPR-Cas9 components if enhancing the safety of gene editing is a concern. A paper by Uchida et al. describes an editing process that uses non-integrating lentiviral vectors (NILVs) to correct the SCD mutation by up to 42% at the protein level.

Do you ever isolate more purified HSPCs (i.e. additional markers to CD34) and perform RNP electroporation on a more purified population?

Danielle Nguyen Truong: The CRISPR gene-editing protocol presented in our webinar has been optimized using bulk CD34⁺ cell editing; it has not been tested on sorted and purified HSPC subsets. However, gene-editing efficiency of HSPC subsets during bulk CD34⁺ cell editing has been evaluated and the data showed efficient editing and maintenance of the CD34⁺CD45RA⁻CD90⁺ HSPC subset. Therefore, the presented genome editing protocol should be applicable when starting with purified HSPC subsets.

Gene editing of more purified HSPCs has been described in the literature. For example, in the paper below, Zonari et al. genome edited sorted CD34⁺CD38⁻ cells and suggested that such an approach may allow for better tailored gene transfer to LT-HSPCs.

Are the pre-editing CRISPR parameters the same as for lentivirus transduction?

Danielle Nguyen Truong: The culture conditions and cell pre-activation of the pre-editing CRISPR is similar as for lentivirus transduction. Please refer to our technical bulletin for instructions on how to culture CD34⁺ cells before transfection.

Do both alleles get edited with the RNP electroporation?

Danielle Nguyen Truong: Both alleles do not necessarily get edited with the RNP electroporation. As non-homologous end joining (NHEJ) is an error-prone DNA repair process, insertions and deletions (indels) are often randomly introduced into the gene, resulting in frameshifts or loss of gene function. It is necessary to determine if there are indels in one or both alleles by subcloning the edited population and sequencing these clones to determine the exact genotype of the edited clones.

If you'd like to confirm successful genome editing prior to transplantation of the cells, can you keep the cells in culture for 2 - 3 days before transplanting? Or is that too long to wait?

Danielle Nguyen Truong: Cells can be grown in culture for an additional 2 or 3 days before transplanting of the cells without any impact to editing efficiency. However, the cells need to be passaged if they are over-confluent to avoid overgrown cultures.

While longer cell culture may be required to confirm successful genome editing, the engraftment potential of the cells decreases with extended cell culture. It has been demonstrated that CD34⁺ cells retain engraftment potential following 6 - 7 days culture in optimal conditions and medium.

Have you used this genome editing system to generate knock-in (KI) cell lines?

Danielle Nguyen Truong: We have demonstrated genetic knock-in with the ArciTect™ CRISPR-Cas9 Genome Editing System, through green fluorescent protein (GFP) to blue fluorescent protein (BFP) conversion, by targeting the enhanced green fluorescent protein (eGFP) locus in eGFP-tagged WLS-1C iPS and H1 ES cell lines. For more details, explore our technical bulletin.

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