Method Article
* These authors contributed equally
The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.
CRISPR/Cas9 is a highly versatile and efficient gene-editing tool adopted widely to correct various genetic mutations. The feasibility of gene manipulation of hematopoietic stem and progenitor cells (HSPCs) in vitro makes HSPCs an ideal target cell for gene therapy. However, HSPCs moderately lose their engraftment and multilineage repopulation potential in ex vivo culture. In the present study, ideal culture conditions are described that improves HSPC engraftment and generate an increased number of gene-modified cells in vivo. The current report displays optimized in vitro culture conditions, including the type of culture media, unique small molecule cocktail supplementation, cytokine concentration, cell culture plates, and culture density. In addition to that, an optimized HSPC gene-editing procedure, along with the validation of the gene-editing events, are provided. For in vivo validation, the gene-edited HSPCs infusion and post-engraftment analysis in mouse recipients are displayed. The results demonstrated that the culture system increased the frequency of functional HSCs in vitro, resulting in robust engraftment of gene-edited cells in vivo.
The inaccessibility to human leukocyte antigen (HLA)-matched donors in allogenic transplantation settings and the rapid development of highly versatile and safe genetic engineering tools make autologous hematopoietic stem cell transplantation (HSCT) a curative treatment strategy for the hereditary blood disorders1,2. Autologous hematopoietic stem and progenitor cell (HSPC) gene therapy involves the collection of patients' HSPCs, genetic manipulation, correction of disease-causing mutations, and transplantation of gene-corrected HSPCs into the patient3,4. However, the successful outcome of the gene therapy relies on the quality of the transplantable gene-modified graft. The gene manipulation steps and ex vivo culture of HSPCs affect the quality of the graft by decreasing the frequency of long-term hematopoietic stem cells (LT-HSCs), necessitating the infusion of large doses of gene-manipulated HSPCs2,5,6.
Several small molecules, including SR1 and UM171, are currently being employed to expand cord blood HSPCs robustly7,8. For adult HSPCs, due to the higher cell yield obtained on mobilization, robust expansion is not required. However, retaining the stemness of isolated HSPCs in ex vivo culture is crucial for its gene therapy applications. Therefore, an approach focusing on the culture enrichment of hematopoietic stem cells (HSCs) is developed using a combination of small molecules: Resveratrol, UM729, and SR1 (RUS)7. The optimized HSPC culture conditions promote the enrichment of HSCs, resulting in increased frequency of gene-modified HSCs in vivo, and reduce the need for gene manipulating large doses of HSPCs, facilitating cost-effective gene therapy approaches8.
Here, a comprehensive protocol for HSPCs culture is described, along with the infusion and analysis of gene-edited cells in vivo.
In vivo experiments on immunodeficient mice were approved by and performed following the guidelines of the Institute Animal Ethics Committee (IAEC), Christian Medical College, Vellore, India. Granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood samples were collected from healthy human donors with informed consent after obtaining Institutional Review Board (IRB) approval.
1. Isolation of peripheral blood mononuclear cells (PBMNCs) and purification of CD34 + cells
2. In vitro culture of purified HSPCs
3. Gene editing of HSPCs
4. Validation of gene-editing events in HSPCs
5. Transplantation of gene-edited HSPCs
6. Assessment of short-term engraftment potential
7. Assessment of long-term engraftment potential
8. Immunophenotyping
9. Assessment of gene-editing frequency in engrafted bone marrow mononuclear cells
The present study identifies ideal HSPC culture conditions that facilitate the retention of CD34+CD133+CD90+ HSCs in ex vivo culture. To demonstrate the culture enrichment of HSCs along with the enhanced generation of gene-modified HSCs, the optimized procedures for PBMNC isolation, CD34+ cell purification, culture, gene editing, transplantation, characterization of engraftment, and gene-modified cells in vivo are provided (Figure 1). Post purification, flow cytometry evaluation was performed to check the HSPC markers, and HSPCs were cultured for 72 h. After 72 h of culture, the HSPCs were nucleofected with Cas9 RNP and cultured for an additional 2 days. The optimized culture conditions containing the RUS cocktail showed increased viability and higher frequency of CD34+CD133+CD90+ HSCs and increased gene-editing frequency (Figure 2). To further demonstrate that the optimized culture conditions increase the frequency of gene-modified cells in vivo, day 3 HSPCs targeting the CCR5 locus were gene-edited and infused into sub-lethally irradiated NSG mice. The engraftment of human cells in mouse bone marrow (BM) was analyzed 16 weeks post infusion (Figure 3A). Flow cytometry analysis of human CD45+ (hCD45) cells in NSG mice showed increased engraftment in the culture conditions (Figure 3B,C). Analysis of the gene-editing frequency in the mouse BM cells showed increased engraftment of gene-edited HSPCs in RUS-supplemented culture conditions (Figure 3D).
Figure 1: Summary of the present study. Graphical summary of the procedure involved in isolating PBMNCs, the magnetic enrichment of CD34+ cells from PBMNCs, culturing, the characterization of human hematopoietic stem and progenitor cells (HSPCs), gene editing, and transplantation is represented. Please click here to view a larger version of this figure.
Figure 2: The RUS cocktail enriches the frequency of HSCs. The mPB-HSPCs were cultured in the stem cell culture media containing cytokines with vehicle (DMSO) and RUS cocktail for 3 days and gene-edited with 25 pM of Cas9-RNP. The gene-edited cells were analyzed by FACS for the markers for HSPCs 48 h post nucleofection. (A) Flow plots represents the live cells (7AAD-) and CD34+ CD90+ population. (B) The percentage and frequency of indel patterns analyzed 72 h post editing in the DMSO and RUS-treated group. (C) The absolute number of CD34+ CD90+ cells analyzed 48 h post editing (n = 2) (donor = 1). (D) The absolute number of total nucleated cells (TNC) analyzed 48 h post-editing (n = 2) (donor = 1). Error bars represent mean ± SEM, *p ≤ 0.05 (unpaired t-test). Please click here to view a larger version of this figure.
Figure 3: Optimized culture conditions increase the frequency of gene-modified cells in vivo. (A) Schematic representation of the experiment. (B) Representative FACS plot showing the hCD45+ cells in the mouse BM. The inset refers to human cells (left) and mouse cells (right). HSPCs were cultured for 3 days, gene-edited with sgRNA on day 3, and transplanted immediately post electroporation. (C) The engraftment of human cells in mouse BM at 16 weeks post infusion (n = 4). (D) The human gene-modified cell (hCD45+ gene-edited cells) chimerism in mouse BM at 16 weeks post-infusion (n = 4) (donor = 1). Each dot represents an individual mouse, and the data points are from an individual experiment. Error bars represent mean ± SEM, *p≤ 0.05 (unpaired t-test). The figure is adapted with permission from Christopher et al.7. Please click here to view a larger version of this figure.
Cell count | Purification buffer (mL) |
< 1 x 107 cells | 0.1 |
1 x 107 - 1 x 108 cells | 0.5 |
1 - 5 x 108 cells | 1 |
Table 1: The volume of purification buffer to prepare the cellular suspension for CD34+ cell purification.
Primer name | Sequence |
CCR5 Forward | CAGAGCCAAGCTCTCCATC |
CCR5 Reverse | AGAGACGCAAACACAGCCA |
CCR5 sequencing Forward primer | AATGTAGACATCTATGTAGG |
Table 2: Primer sequence for amplifying the CCR5 locus.
Components | 50 µL reaction |
Buffer (5x) | 10 µL |
Forward primer (10 µM) | 1 µL |
Reverse primer (10 µM) | 1 µL |
dNTP | 4 µL |
Polymerase | 1 µL |
Genomic DNA | 200 ng |
Nuclease free water | upto 50 µL |
Table 3: The reaction mixture for amplifying the CCR5 locus using PCR.
Steps | Duration | Temperature | No of Cycles |
Initial denaturation | 1 min | 95 °C | 1 |
Denaturation | 10 s | 98 °C | 35 |
Annealing | 15 s | 56 °C | |
Extension | 30 s | 68 °C | |
Final extension | 1 min | 72 °C | 1 |
Hold | ∞ | 15 °C |
Table 4: Thermocycler conditions for amplifying the CCR5 locus.
Components | 10 µL reaction |
Buffer (5x) | 2 µL |
primer (2 µM) | 1.6 µL |
RR mix | 0.75 µL |
PCR cleanup product | 80 ng |
Nuclease free water | upto 10 µL |
Table 5: The reaction mixture for Sanger sequencing PCR.
Steps | Duration | Temperature | No of cycles |
Denaturation | 15 s | 96 °C | 27 |
Annealing | 20 s | 55 °C | |
Extension | 4 min | 60 °C | |
Hold | ∞ | 15 °C |
Table 6: Thermocycler conditions for Sanger sequencing PCR.
Buffer | Composition |
10x RBC lysis buffer – 100 mL (pH – 7.3) | 8.26 g of NH4Cl, 1.19 g of NaHCO3, 200 µL of EDTA (0.5 M, pH8) |
50x TAE buffer (pH – 8.3) | Dissolve 50 mM EDTA sodium salt, 2 M Tris, 1 M glacial acetic acid in 1 L of water |
Table 7: Buffer Compositions
Antibodies | Volume |
Anti-human CD45 APC | 3 µL |
Anti-mouse CD45.1 PerCP-Cy5 | 4.5 µL |
Anti-mouse CD34 PE | 3 µL |
Table 8: Antibodies used for assessing human cell engraftment.
Antibodies | Volume |
Anti-human CD45 APC | 3 µL |
Anti-mouse CD19 PerCP | 15 µL |
Anti-mouse CD13 PE | 15 µL |
Anti-mouse CD3 PE-Cy7 | 2 µL |
Table 9: Antibodies used for assessing the proportion of multilineage cells derived from engrafted HSPCs.
The successful outcome of HSPC gene therapy relies predominantly on the quality and quantity of engraftable HSCs in the graft. However, the functional properties of HSCs are highly affected during the preparatory phase of gene therapy products, including by in vitro culture and toxicity associated with the gene manipulation procedure. To overcome these limitations, we have identified ideal HSPCs culture conditions that retain the stemness of CD34+CD133+CD90+ HSCs in ex vivo culture. Many research groups have used SR1 or UM171 or other molecules as stand-alone molecules to expand umbilical cord blood (UCB) HSPCs in vitro23,24. A previous study used a combination of both SR1 and UM17125. The small molecules and cytokines of the culture medium were specifically optimized for mobilized adult HSPCs and their application in autologous gene therapy. The screening experiment showed that combining three small molecules Resveratrol, UM729, and SR1, is important for generating a high number of CD34+CD90+ cells and inhibiting the proliferation of differentiated and committed progenitor cells. The UM729 in the RUS cocktail can be replaced with UM171. However, the commercial procurement of UM171 is less feasible. The cytokine concentrations are adopted from the protocol that was employed in clinical studies26 to reduce the variabilities during the scale-up process. The cytokine cocktail contains IL6 instead of IL3 to minimize progenitor proliferation and HSC exhaustion in vitro27. Preparing fresh aliquots of the culture media (basal media + RUS + cytokine cocktails) is recommended to reduce experimental variation and obtain high reproducibility. The protocol is applicable for both NHEJ- and HDR-mediated gene editing. In particular, HSPC culture of 48-72 h before electroporation and 24 h post electroporation is crucial for HDR gene editing. The optimized culture conditions should benefit HDR gene editing by preserving the stem cells. It was also observed that the culture conditions assist lentiviral transduction in long-term HSCs. This suggests that, if viral particles like AAV6 or IDLV are used as an HDR donor, the HDR editing efficiency is expected to improve, as the optimized culture conditions promote donor delivery into HSCs.
To assess gene editing outcomes, including NHEJ and HDR, NGS analysis, probe or ddPCR analysis, or Sanger sequencing7,8,28 is suggested, followed by deconvolution using online tools (ICE/ICE Knock-In)16, owing to its robust quantitative nature. Alternatively, a T7 endonuclease assay can be performed on edited DNA samples, and the fragmented DNA bands can be quantified using ImageJ. However, the T7 endonuclease assay approach is less precise than deconvolution analysis and targets next-generation sequencing.
The transplantation protocol is also optimized by conditioning the NBSGW mice with busulfan, enabling low cell doses to assess HSPC engraftment and repopulation. Overall, this procedure must reduce the doses of HSPCs required for gene manipulation and increase the accessibility of HSPC gene therapy in developing countries.
In the present study, the protocols for PBMNC isolation, CD34+ HSPC purification, gene editing and validation, and assessment of the engrafted gene-edited HSPCs in mouse bone marrow were demonstrated. It has also been proven that the optimized HSPC culture enriches CD34+CD133+CD90+ HSPCs and increases the chimerism of gene-edited cells in vivo.
The authors declare that no competing financial interests exist.
The authors want to acknowledge the staff of the flow cytometry facility and animal facility of CSCR. A. C. is funded by an ICMR-SRF fellowship, K. V. K. is funded by a DST-INSPIRE fellowship, and P. B. is funded by a CSIR-JRF fellowship. This work was funded by the Department of Biotechnology, Government of India (grant no. BT/PR26901/MED/31/377/2017 and BT/PR31616/MED/31/408/2019)
Name | Company | Catalog Number | Comments |
4D-Nucleofector® X Unit | LONZA BIOSCIENCE | AAF-1003X | |
4D-Nucleofector™ X Kit ( 16-well Nucleocuvette™ Strips) | LONZA BIOSCIENCE | V4XP-3032 | |
Antibiotic-Antimycotic (100X) | THERMO SCIENTIFIC | 15240096 | |
Anti-human CD45 APC | BD BIOSCIENCE | 555485 | |
Anti-human CD13 PE | BD BIOSCIENCE | 555394 | |
Anti-human CD19 PerCP | BD BIOSCIENCE | 340421 | |
Anti-human CD3 PE-Cy7 | BD BIOSCIENCE | 557749 | |
Anti-human CD90 APC | BD BIOSCIENCE | 561971 | |
Anti-human CD133/1 | Miltenyibiotec | 130-113-673 | |
Anti-human CD34 PE | BD BIOSCIENCE | 348057 | |
Anti-mouse CD45.1 PerCP-Cy5 | BD BIOSCIENCE | 560580 | |
Blood Irradator-2000 | BRIT (Department of Biotechnology, India) | BI 2000 | |
Cell culture dish (delta surface-treated 6-well plates) | NUNC (THERMO SCIENTIFIC) | 140675 | |
CrysoStor CS10 | BioLife solutions | #07952 | |
Busulfan | CELON LABS (60mg/10mL) | - | |
Guide-it Recombinant Cas9 | TAKARA BIO | 632640 | |
Cas9-eGFP | SIGMA | C120040 | |
Centrifuge tube-15ml | CORNING | 430790 | |
Centrifuge tube-50ml | NUNC (THERMO SCIENTIFIC) | 339652 | |
DMSO | MPBIO | 219605590 | |
DNAase | STEMCELL TECHNOLOGIES | 6469 | |
Dulbecco′s Phosphate Buffered Saline- 1X | HYCLONE | SH30028.02 | |
EasySep™ Human CD34 Positive Selection Kit II | STEMCELL TECHNOLOGIES | 17856 | |
EasySep magnet | STEMCELL TECHNOLOGIES | 18000 | |
Electrophoresis unit | ORANGE INDIA | HDS0036 | |
FBS | THERMO SCIENTIFIC | 10270106 | |
Flow cytometer – ARIA III | BD BIOSCIENCE | - | |
FlowJo | BD BIOSCIENCE | - | |
Flt3-L | PEPROTECH | 300-19-1000 | |
Gel imaging system | CELL BIOSCIENCES | 11630453 | |
HighPrep DTR reagent | MAGBIOGENOMICS | DT-70005 | |
Human BD Fc Block | BD BIOSCIENCE | 553141 | |
IL6 | PEPROTECH | 200-06-50 | |
IMDM media | THERMO SCIENTIFIC | 12440053 | |
Infrared lamp | MURPHY | - | |
Insulin syringe 6mm 31G | BD BIOSCIENCE | 324903 | |
Ketamine | KETMIN 50 | - | |
Loading dye 6X | TAKARA BIO | 9156 | |
Lymphoprep | STEMCELL TECHNOLOGIES | 7851 | |
Mice Restrainer | AVANTOR | TV-150 | |
Nano drop spectrophotometer | THERMO SCIENTIFIC | ND-2000C | |
Neubauer cell counting chamber | ROHEM INSTRUMENTS | CC-3073 | |
NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) | The Jackson Laboratory | RRID:IMSR_JAX:005557 | |
NOD,B6.SCID Il2rγ−/−KitW41/W41 (NBSGW) | The Jackson Laboratory | RRID:IMSR_JAX:026622 | |
Nunc delta 6-well plate | THERMO SCIENTIFIC | 140675 | |
Polystyrene round-bottom tube | BD | 352008 | |
P3 primary cell Nucleofection solution | LONZA BIOSCIENCE | PBP3-02250 | |
Pasteur pipette | FISHER SCIENTIFIC | 13-678-20A | |
PCR clean-up kit | TAKARA BIO | 740609.25 | |
Mouse Pie Cage | FISCHER SCIENTIFIC | 50-195-5140 | |
polystyrene round-bottom tube (12 x 75 mm) | STEMCELL TECHNOLOGIES | 38007 | |
Primer3 | Whitehead Institute for Biomedical Research | https://primer3.ut.ee/ | |
QuickExtract™ DNA Extraction Solution | Lucigen | QE09050 | |
Reserveratrol | STEMCELL TECHNOLOGIES | 72862 | |
SCF | PEPROTECH | 300-07-1000 | |
SFEM-II | STEMCELL TECHNOLOGIES | 9655 | |
sgRNA | SYNTHEGO | - | |
SPINWIN | TARSON | 1020 | |
StemReginin 1 | STEMCELL TECHNOLOGIES | 72342 | |
ICE analysis tool | SYNTHEGO | https://ice.synthego.com/ | |
Tris-EDTA buffer solution (TE) 1X | SYNTHEGO | Supplied with gRNA | |
Thermocycler | APPLIED BIOSYSTEMS | 4375305 | |
TPO | PEPROTECH | 300-18-1000 | |
Trypan blue | HIMEDIA LABS | TCL046 | |
UM171 | STEMCELL TECHNOLOGIES | 72914 | |
UM729 | STEMCELL TECHNOLOGIES | 72332 | |
Xylazine | XYLAXIN - INDIAN IMMUNOLOGICALS LIMITED | - |
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