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)

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 <su...

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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&#160;v...

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&#39; 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >4D-Nucleofector&#174; X Unit</td>
			<td >LONZA BIOSCIENCE</td>
			<td >AAF-1003X</td>
			<td ></td>
		</tr>
		<tr >
			<td >4D-Nucleofector&#8482; X Kit ( 16-well Nucleocuvette&#8482; Strips)</td>
			<td >LONZA BIOSCIENCE</td>
			<td >V4XP-3032</td>
			<td></td>
		</tr>
		<tr >
			<td >Antibiotic-Antimycotic (100X)</td>
			<td >THERMO SCIENTIFIC</td>
			<td >15240096</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD45 APC</td>
			<td >BD BIOSCIENCE&#160;</td>
			<td >555485&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD13 PE</td>
			<td >BD BIOSCIENCE</td>
			<td >555394</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD19 PerCP</td>
			<td >BD BIOSCIENCE</td>
			<td >340421</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD3 PE-Cy7</td>
			<td >BD BIOSCIENCE</td>
			<td >557749</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD90 APC</td>
			<td >BD BIOSCIENCE</td>
			<td>561971</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD133/1&#160;</td>
			<td >Miltenyibiotec</td>
			<td >130-113-673</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-human CD34 PE</td>
			<td >BD BIOSCIENCE</td>
			<td >348057</td>
			<td></td>
		</tr>
		<tr >
			<td >Anti-mouse CD45.1 PerCP-Cy5</td>
			<td >BD BIOSCIENCE</td>
			<td >560580</td>
			<td></td>
		</tr>
		<tr >
			<td >Blood Irradator-2000</td>
			<td >&#160;BRIT (Department of Biotechnology, India)</td>
			<td >BI 2000&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell culture dish (delta surface-treated 6-well plates)</td>
			<td >NUNC (THERMO SCIENTIFIC)</td>
			<td >140675</td>
			<td></td>
		</tr>
		<tr >
			<td >CrysoStor CS10</td>
			<td >BioLife solutions</td>
			<td >#07952</td>
			<td></td>
		</tr>
		<tr >
			<td >Busulfan</td>
			<td >CELON LABS (60mg/10mL)</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Guide-it Recombinant Cas9</td>
			<td >TAKARA BIO</td>
			<td >632640</td>
			<td></td>
		</tr>
		<tr >
			<td >Cas9-eGFP</td>
			<td >SIGMA</td>
			<td >C120040&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;Centrifuge tube-15ml</td>
			<td >CORNING</td>
			<td >430790</td>
			<td></td>
		</tr>
		<tr >
			<td >&#160;Centrifuge tube-50ml</td>
			<td >NUNC (THERMO SCIENTIFIC)</td>
			<td >339652</td>
			<td></td>
		</tr>
		<tr >
			<td >DMSO</td>
			<td >MPBIO</td>
			<td >219605590</td>
			<td></td>
		</tr>
		<tr >
			<td >DNAase</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >6469</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#8242;s Phosphate Buffered Saline- 1X</td>
			<td >HYCLONE</td>
			<td >SH30028.02</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep&#8482; Human CD34 Positive Selection Kit II</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td>17856</td>
			<td></td>
		</tr>
		<tr >
			<td >EasySep magnet</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >18000</td>
			<td></td>
		</tr>
		<tr >
			<td >Electrophoresis unit</td>
			<td >ORANGE INDIA</td>
			<td >HDS0036</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS</td>
			<td >THERMO SCIENTIFIC</td>
			<td >10270106</td>
			<td></td>
		</tr>
		<tr >
			<td >Flow cytometer &#8211; ARIA III</td>
			<td >BD BIOSCIENCE</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >FlowJo&#160;</td>
			<td >BD BIOSCIENCE</td>
			<td >&#160;-</td>
			<td></td>
		</tr>
		<tr >
			<td >Flt3-L</td>
			<td >PEPROTECH</td>
			<td >300-19-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Gel imaging system</td>
			<td >CELL BIOSCIENCES</td>
			<td >11630453</td>
			<td></td>
		</tr>
		<tr >
			<td >HighPrep DTR reagent</td>
			<td >MAGBIOGENOMICS</td>
			<td >DT-70005</td>
			<td></td>
		</tr>
		<tr >
			<td >Human BD Fc Block</td>
			<td >BD BIOSCIENCE</td>
			<td >553141</td>
			<td></td>
		</tr>
		<tr >
			<td >IL6</td>
			<td >PEPROTECH</td>
			<td >200-06-50</td>
			<td></td>
		</tr>
		<tr >
			<td >IMDM media</td>
			<td >THERMO SCIENTIFIC</td>
			<td >12440053</td>
			<td></td>
		</tr>
		<tr >
			<td >Infrared lamp</td>
			<td >MURPHY</td>
			<td >-</td>
			<td></td>
		</tr>
		<tr >
			<td >Insulin syringe 6mm 31G</td>
			<td >BD BIOSCIENCE</td>
			<td >324903</td>
			<td></td>
		</tr>
		<tr >
			<td >Ketamine</td>
			<td >KETMIN 50</td>
			<td >-</td>
			<td></td>
		</tr>
		<tr >
			<td >Loading dye 6X</td>
			<td >TAKARA BIO</td>
			<td >9156</td>
			<td></td>
		</tr>
		<tr >
			<td >Lymphoprep</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >7851</td>
			<td></td>
		</tr>
		<tr >
			<td >Mice Restrainer</td>
			<td >AVANTOR</td>
			<td >TV-150</td>
			<td></td>
		</tr>
		<tr >
			<td >Nano drop spectrophotometer</td>
			<td >THERMO SCIENTIFIC</td>
			<td >ND-2000C</td>
			<td></td>
		</tr>
		<tr >
			<td >Neubauer cell counting chamber</td>
			<td >ROHEM INSTRUMENTS</td>
			<td >CC-3073</td>
			<td></td>
		</tr>
		<tr >
			<td >NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG)</td>
			<td >The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:005557</td>
			<td></td>
		</tr>
		<tr >
			<td >NOD,B6.SCID Il2r&#947;&#8722;/&#8722;KitW41/W41 (NBSGW)</td>
			<td >The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:026622</td>
			<td></td>
		</tr>
		<tr >
			<td >Nunc delta 6-well plate</td>
			<td >THERMO SCIENTIFIC</td>
			<td >140675</td>
			<td></td>
		</tr>
		<tr >
			<td >Polystyrene round-bottom tube</td>
			<td >BD</td>
			<td >352008</td>
			<td></td>
		</tr>
		<tr >
			<td >P3 primary cell Nucleofection solution</td>
			<td >LONZA BIOSCIENCE</td>
			<td >PBP3-02250</td>
			<td></td>
		</tr>
		<tr >
			<td >Pasteur pipette</td>
			<td >FISHER SCIENTIFIC</td>
			<td >13-678-20A</td>
			<td></td>
		</tr>
		<tr >
			<td >PCR clean-up kit</td>
			<td >TAKARA BIO</td>
			<td >740609.25</td>
			<td></td>
		</tr>
		<tr >
			<td >Mouse Pie Cage</td>
			<td>FISCHER SCIENTIFIC</td>
			<td>50-195-5140</td>
			<td></td>
		</tr>
		<tr >
			<td >polystyrene round-bottom tube (12 x 75 mm)</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >38007</td>
			<td></td>
		</tr>
		<tr >
			<td >Primer3</td>
			<td >Whitehead Institute for Biomedical Research</td>
			<td >https://primer3.ut.ee/</td>
			<td></td>
		</tr>
		<tr >
			<td >QuickExtract&#8482; DNA Extraction Solution</td>
			<td >Lucigen</td>
			<td >QE09050</td>
			<td></td>
		</tr>
		<tr >
			<td >Reserveratrol</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72862</td>
			<td></td>
		</tr>
		<tr >
			<td >SCF</td>
			<td >PEPROTECH</td>
			<td >300-07-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >SFEM-II</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >9655</td>
			<td></td>
		</tr>
		<tr >
			<td >sgRNA</td>
			<td >SYNTHEGO</td>
			<td >-&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >SPINWIN</td>
			<td >TARSON</td>
			<td >1020</td>
			<td></td>
		</tr>
		<tr >
			<td >StemReginin 1</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72342</td>
			<td></td>
		</tr>
		<tr >
			<td >ICE analysis tool</td>
			<td >SYNTHEGO</td>
			<td >https://ice.synthego.com/</td>
			<td></td>
		</tr>
		<tr >
			<td >Tris-EDTA buffer solution (TE) 1X</td>
			<td >SYNTHEGO</td>
			<td >Supplied with gRNA&#160;</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermocycler</td>
			<td >APPLIED BIOSYSTEMS</td>
			<td >4375305</td>
			<td></td>
		</tr>
		<tr >
			<td >TPO</td>
			<td >PEPROTECH</td>
			<td >300-18-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Trypan blue</td>
			<td >HIMEDIA LABS</td>
			<td >TCL046</td>
			<td></td>
		</tr>
		<tr >
			<td >UM171</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72914</td>
			<td></td>
		</tr>
		<tr >
			<td >UM729</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72332</td>
			<td></td>
		</tr>
		<tr >
			<td >Xylazine</td>
			<td >XYLAXIN - INDIAN IMMUNOLOGICALS LIMITED</td>
			<td >-</td>
			<td></td>
		</tr>
	</tbody>
</table>

crispr/cas9 gene editing of hematopoietic stem and progenitor cells for gene therapy applications

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 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</stron...

Watch this Scientific Journal Video about CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications at JoVE.com

CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

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 ...

Ex vivo culture can hamper the repopulation potential of hematopoietic stem and progenitor cells. Our protocol preserves the stemness during the culture and thus improves the frequency of gene-modified cells in vivo. Hematopoietic stem cell gene therapy is currently being employed for monogenic diseases and infectious disease like HIV.The HSPC gene therapy protocol heavily relies on ex vivo culture. Our protocol should be compatible with any procedure that employs ex vivo culture. Demonstrating this protocol will be Vigneshwaran Venkatesan, PhD student from my laboratory.After isolating the HSPCs, culture them in stem cell culture media that contains cytokines and small molecules Resveratrol, UM729, and StemRegenin1. Before gene editing, set the ThermoMixer at 37 degrees Celsius to reconstitute the guide RNA, then preheat the TE buffer at 37 degrees Celsius for 10 minutes. Centrifuge the synthetic chemically-modified sgRNA vial at 11, 000 times g for 1 minute at 4 degrees Celsius and add 15 microliters of TE buffer to the vial containing 1.5 nanomolar of lyophilized sgRNA and mix by gentle swirling to get a final concentration of 100 picomolar per microliter.Incubate the vial in the ThermoMixer with minimal shaking at 37 degrees Celsius for 30 to 40 seconds. Gently tap the vial on the sides and spin the vial to collect 15 microliters of sgRNA. Make 1-microliter aliquots and store them at minus 80 degrees Celsius for up to 1 year.For nucleofection, pellet 2 times 10 to the 5th cells by centrifuging at 200 times g for 5 minutes at room temperature. Once the supernatant is discarded, resuspend the pellet in 20 microliters of the buffer. Gently mix the cell suspension with the prepared RNP complex avoiding air bubbles.Now, transfer the suspension to the commercial nucleofection strip and electroporate the cells using the 4D-Nucleofector by selecting the pulse code DZ-100. To the electroporated cells in the nucleofection strip, add 100 microliters of pre-incubated culture media and leave the cells undisturbed for 10 minutes at room temperature. At the end of the incubation, transfer the contents to the culture plate as per the experimental requirements.Place the NSG mice in the pie cages in 6 to 8 hours prior to HSPC transplantation. Irradiate the mice at 3.5 gray using a commercially-available irradiator. For NBSGW, pre-condition 6 to 8-week-old male and female mice by intraperitoneally injecting busulfan at a dose of 12.5 milligrams per kilogram of body weight 48 hours before HSPC transplantation.For infusing 1 mouse, pellet 6 times 10 to the 5th cells in a 1.5-milliliter tube by centrifuging at 200 times g for 5 minutes. at room temperature. Gently pipette out the supernatant without disturbing the pellet and resuspend the cell pellet in 100 microliters of PBS.Place the pre-conditioned NSG or NBSGW mice in the mouse restrainer and infuse the HSPCs by tail vein injection. Hold the mouse tail and gently push the plug to restrain the mouse. Gently wipe the mouse tail with 70%ethanol.Using a 31 gauge insulin syringe, aspirate 100 microliters of the cell suspension. Now, direct the light from the infrared lamp onto the tail for 30 to 40 seconds, covering the body area of the mouse with folds of tissue paper. To maintain the tail in the planer axis with the syringe, lift it with the left index finger.Gently insert the bevel part of the needle into the left or right caudal tail vein at an angle of 20 degrees. Push the plunger to infuse the cell suspension into the vein and apply gentle pressure near the pierced region with tissue paper and pull out the needle. After anesthetizing, position the animal in ventral recumbency and gently scruff the mice to open the eye, allowing the eye's globe to protrude slightly.Gently insert the pasture pipette at a 30 to 45 degree angle into the medial canthus of the eye under the nictating membrane. After placing the Pasteur pipette at the proper position, apply slight pressure to the tube and begin to rotate the tube gently. After collecting 50 to 80 microliters of peripheral blood, gently withdraw the pipette from the medial canthus of the eye.Post-euthanization, make a vertical incision 1 centimeter above the urethra and extend until 1 centimeter below the diaphragm. Cut horizontally at the incised area's corners to open the abdominal region. After dissecting the femur and tibia, use scissors to remove the soft tissues attached to the femur and tibia and gently scrub the bones with a tissue paper.Use a scalpel to make a small hole with a diameter not more than 0.2 centimeters at the bottom of a 0.5-milliliter microcentrifuge tube. Remove the proximal ends of the bones using a scalpel and place the bones with the cut side facing toward the hole of the 0.5-milliliter micro centrifugation tube. Now, place the 0.5-milliliter tube with the bones in the 1.5-milliliter tube containing 100 microliters of sterile PBS.Close the lid, spin the tubes at 200 times g for 3 minutes under sterile conditions at room temperature and discard the 0.5-milliliter tubes containing bones with an empty marrow cavity. To the 1.5-milliliter reaction tube containing bone marrow, add 1 milliliter of PBS, then using a 1-milliliter pipette, gently resuspend the cells 10 times. Transfer 1 milliliter of the cell suspension to a 15-milliliter tube containing 9 milliliters of RBC lysis buffer.Incubate the cells in ice for 7 minutes by gently inverting the tube every two minutes. After incubation, centrifuge the tube at 200 times g for 5 minutes at room temperature and repeat the washing until a clear pale white pellet is observed. The frequency of live cells and CD34-positive, CD90-positive cells have increased in the RUS group after 48 hours of nucleofection.After 72 hours, the percentage and frequency of indel increased in the RUS-treated group as compared to DMSO-treated group suggesting an increase in gene editing. The absolute number of CD34-positive, CD90-positive cells, and total nucleated cells are significantly higher 48 hours post-editing. Flow cytometry analysis of human CD45-positive cells in the NSG mice showed increased engraftment in the culture conditions.Analysis of the gene-editing frequency in the mouse BM cells showed increased engraftment of gene-edited HSPCs in RUS-supplemented culture conditions. Culturing the HSPCs at a confluence of 200, 000 cells per mL is crucial to improve the functional stem cells. Amplifying the cord blood stem cells and the iSPC-derived stem cells are the new areas in which this protocol is being tested.

crisprcas9-gene-editing-hematopoietic-stem-progenitor-cells-for-gene

Research

JoVE Journal

Biology

3.2K ビュー.  Christian Medical College campus. 生体外培養は、造血幹および前駆細胞の再増殖の可能性を妨げる可能性があります。私たちのプロトコルは、培養中に幹細胞性を維持し、したがって、in vivoでの遺伝子改変細胞の頻度を改善します。造血幹細胞遺伝子治療は現在、単一遺伝子疾患やHIVなどの感染症に採用されています。HSPC遺伝子治療プロトコルは、生体外培養に大きく依存しています。私たちの?...

ビデオ: CRISPR/Cas9 Gene Editing of Hematopoietic Stem and Progenitor Cells for Gene Therapy Applications

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering na&iuml;ve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated <a href="http://www.gudmap.org/">http://www.gudmap.org/</a>, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which greatly facilitates the study of candidate stem/progenitor cell regulator effects. Basic cell biological parameters such as proliferation and cell death as well as changes in expression of molecular markers characteristic of nephron stem/progenitor cells in vivo can be successfully used as experimental outcomes. Ongoing work in our laboratory using this novel primary cell technique aims to uncover basic mechanisms governing the regulation of self-renewal versus differentiation in nephron stem/progenitor cells.

In this report we describe a method for the isolation and culture of the progenitor cell niche from the embryonic mouse kidney that can be used to study signaling pathways regulating stem/progenitor cells of the developing kidney. These cultured cells are highly accessible to small molecule and recombinant protein treatment, and importantly also to viral transduction, which allows efficient manipulation of candidate pathways.

Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and  to gain ...

Induction and Testing of Hypoxia in Cell Culture

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells. This decrease of oxygen tension can be due to a reduced supply in oxygen (causes include insufficient blood vessel network, defective blood vessel, and anemia) or to an increased consumption of oxygen relative to the supply (caused by a sudden higher cell proliferation rate). Hypoxia can be physiologic or pathologic such as in solid cancers 1-3, rheumatoid arthritis, atherosclerosis etc&#x2026; Each tissues and cells have a different ability to adapt to this new condition. During hypoxia, hypoxia inducible factor alpha (HIF) is stabilized and regulates various genes such as those involved in angiogenesis or transport of oxygen 4. The stabilization of this protein is a hallmark of hypoxia, therefore detecting HIF is routinely used to screen for hypoxia 5-7. 
In this article, we propose two simple methods to induce hypoxia in mammalian cell cultures and simple tests to evaluate the hypoxic status of these cells.

Here we propose simple methods to induce hypoxia in cell cultures and simple tests to evaluate the hypoxic status of the cultures.

Hypoxia is defined as the reduction or lack of oxygen in organs, tissues, or cells.  This decrease of oxygen tension can be due to a reduced supply in ...

Metabolic Labeling of Leucine Rich Repeat Kinases 1 and 2 with Radioactive Phosphate

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, a Ras of complex proteins domain (ROC), a C-terminal of ROC domain (COR), and leucine-rich and ankyrin-like repeats at the N-terminus. The precise cellular roles of LRRK1 and LRRK2 have yet to be elucidated, however LRRK1 has been implicated in tyrosine kinase receptor signaling1,2, while LRRK2 is implicated in the pathogenesis of Parkinson's disease3,4. In this report, we present a protocol to label the LRRK1 and LRRK2 proteins in cells with 32P orthophosphate, thereby providing a means to measure the overall phosphorylation levels of these 2 proteins in cells. In brief, affinity tagged LRRK proteins are expressed in HEK293T cells which are exposed to medium containing 32P-orthophosphate. The 32P-orthophosphate is assimilated by the cells after only a few hours of incubation and all molecules in the cell containing phosphates are thereby radioactively labeled. Via the affinity tag (3xflag) the LRRK proteins are isolated from other cellular components by immunoprecipitation. Immunoprecipitates are then separated via SDS-PAGE, blotted to PVDF membranes and analysis of the incorporated phosphates is performed by autoradiography (32P signal) and western detection (protein signal) of the proteins on the blots. The protocol can readily be adapted to monitor phosphorylation of any other protein that can be expressed in cells and isolated by immunoprecipitation.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are multidomain proteins which encode both GTPase and kinase domains and which are phosphorylated in cells. Here, we present a protocol to label LRRK1 and LRRK2 in cells with 32P orthophosphate, thereby providing a means to measure their overall cellular phophorylation levels.

Leucine rich repeat kinases 1 and 2 (LRRK1 and LRRK2) are paralogs which share a similar domain organization, including a serine-threonine kinase domain, ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Silencing the Spark: CRISPR/Cas9 Genome Editing in Weakly Electric Fish

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these independently derived phenotypes, which share a common genetic architecture. This is perhaps best exemplified by the numerous convergent features of gymnotiforms and mormyrids, two species-rich teleost clades that produce and detect weak electric fields and are called weakly electric fish. In the 50 years since the discovery that weakly electric fish use electricity to sense their surroundings and communicate, a growing community of scientists has gained tremendous insights into evolution of development, systems and circuits neuroscience, cellular physiology, ecology, evolutionary biology, and behavior. More recently, there has been a proliferation of genomic resources for electric fish. Use of these resources has already facilitated important insights with regards to the connection between genotype and phenotype in these species. A major obstacle to integrating genomics data with phenotypic data of weakly electric fish is a present lack of functional genomics tools. We report here a full protocol for performing CRISPR/Cas9 mutagenesis that utilizes endogenous DNA repair mechanisms in weakly electric fish. We demonstrate that this protocol is equally effective in both the mormyrid species Brienomyrus brachyistius and the gymnotiform Brachyhypopomus gauderio by using CRISPR/Cas9 to target indels and point mutations in the first exon of the sodium channel gene scn4aa. Using this protocol, embryos from both species were obtained and genotyped to confirm that the predicted mutations in the first exon of the sodium channel scn4aa were present. The knock-out success phenotype was confirmed with recordings showing reduced electric organ discharge amplitudes when compared to uninjected size-matched controls.

Here, a protocol is presented to produce and rear CRISPR/Cas9 genome knockout electric fish. Outlined in detail are the required molecular biology, breeding, and husbandry requirements for both a gymnotiform and a mormyrid, and injection techniques to produce Cas9-induced indel F0 larvae.

Electroreception and electrogenesis have changed in the evolutionary history of vertebrates. There is a striking degree of convergence in these ...

TGF-&#946;-mediated Endothelial to Mesenchymal Transition (EndMT) and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like phenotype, a process that is termed endothelial to mesenchymal transition (EndMT). Emerging results have suggested that EndMT is causally linked to multiple human diseases, such as fibrosis and cancer. In addition, endothelial-derived mesenchymal cells may be applied in tissue regeneration procedures, as they can be further differentiated into various cell types (e.g., osteoblasts and chondrocytes). Thus, the selective manipulation of EndMT may have clinical potential. Like epithelial-mesenchymal transition (EMT), EndMT can be strongly induced by the secreted cytokine transforming growth factor-beta (TGF-&#946;), which stimulates the expression of so-called EndMT transcription factors (EndMT-TFs), including Snail and Slug. These EndMT-TFs then up- and downregulate the levels of mesenchymal and endothelial proteins, respectively. Here, we describe methods to investigate TGF-&#946;-induced EndMT in vitro, including a protocol to study the role of particular TFs in TGF-&#946;-induced EndMT. Using these techniques, we provide evidence that TGF-&#946;2 stimulates EndMT in murine pancreatic microvascular endothelial cells (MS-1 cells), and that the genetic depletion of Snail using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated gene editing, abrogates this phenomenon. This approach may serve as a model to interrogate potential modulators of endothelial biology, and can be used to perform genetic or pharmacological screens in order to identify novel regulators of EndMT, with potential application in human disease.

TGF-&amp;#946;-mediated Endothelial to Mesenchymal Transition EndMT and the Functional Assessment of EndMT Effectors using CRISPR/Cas9 Gene Editing

We describe methods to investigate TGF-&#946;2-induced EndMT in endothelial cells by observing cell morphology changes and examining the expression EndMT-related marker changes using immunofluorescence staining. CRISPR/Cas9 gene editing was described and used to deplete the gene encoding Snail to investigate its role in TGF-&#946;2-induced EndMT.

In response to specific external cues and the activation of certain transcription factors, endothelial cells can differentiate into a mesenchymal-like ...

Three-Dimensional, Serum-Free Culture System for Lacrimal Gland Stem Cells

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture method to obtain a sufficient number of LG stem cells (LGSCs) is one obstacle for further research and application. The three-dimensional (3D), serum-free culture method for adult mouse LGSCs is well established and shown here. The LGSCs could be continuously passaged and induced to differentiate to acinar or ductal-like cells.
For the LGSC primary culture, the LGs from 6-8-week-old mice were digested with dispase, collagenase I, and trypsin-EDTA. A total of 1 &#215; 104 single cells were seeded into 80 &#181;L of matrix gel-lacrimal gland stem cell medium (LGSCM) matrix in each well of a 24-well plate, precoated with 20 &#181;L of matrix gel-LGSCM matrix. The mix was solidified after incubation for 20 min at 37 &#176;C, and 600 &#181;L of LGSCM added.
For LGSC maintenance, LGSCs cultured for 7 days were disaggregated into single cells by dispase and trypsin-EDTA. The single cells were implanted and cultured according to the method used in the LGSC primary culture. LGSCs could be passaged over 40 times and continuously express stem/progenitor cell markers Krt14, Krt5, P63, and nestin. LGSCs cultured in LGSCM have self-renewal capacity and can differentiate into acinar or ductal-like cells in vitro and in vivo.

The three-dimensional, serum-free culture method for adult lacrimal gland (LG) stem cells is well established for the induction of LG organoid formation and differentiation into acinar or ductal-like cells.

Lacrimal gland (LG) stem cell-based therapy is a promising strategy for lacrimal gland diseases. However, the lack of a reliable, serum-free culture ...

CRISPR Gene Editing Tool for MicroRNA Cluster Network Analysis

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published studies focus on a single miRNA when characterizing the role of small RNAs in cancer. However, ~30%&#160;of human miRNA genes are organized in clustered units that are often co-expressed, indicating a complex and coordinated system of noncoding RNA regulation. A clearer understating of how clustered miRNA networks function cooperatively to regulate tumor growth, cancer aggressiveness, and drug resistance is required before translating noncoding small RNAs to the clinic.
The use of a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR)-mediated gene editing procedure has been employed to study the oncogenic role of a genomic cluster of seven miRNA genes located within a locus spanning ~35,000 bp in length in the context of prostate cancer. For this approach, human cancer cell lines were infected with a lentivirus vector for doxycycline (DOX)-inducible Cas9 nuclease grown in DOX-containing medium for 48 h. The cells were subsequently co-transfected with synthetic trans-activating CRISPR RNA (tracrRNA) complexed with genomic site-specific CRISPR RNA (crRNA) oligonucleotides to allow the rapid generation of cancer cell lines carrying the entire miRNA cluster deletion and individual or combination miRNA gene cluster deletions within a single experiment.
The advantages of this high-throughput gene editing system are the ability to avoid time-consuming DNA vector subcloning, the flexibility in transfecting cells with unique guide RNA combinations in a 24-well format, and the lower-cost PCR genotyping using crude cell lysates. Studies using this streamlined approach promise to uncover functional redundancies and synergistic/antagonistic interactions between miRNA cluster members, which will aid in characterizing the complex small noncoding RNA networks involved in human disease and better inform future therapeutic design.

This protocol describes a high-throughput clustered regularly interspaced short palindromic repeats (CRISPR) gene editing workflow for microRNA cluster network analysis that allows the rapid generation of a panel of genetically modified cell lines carrying unique miRNA cluster member deletion combinations as large as 35 kb within a single experiment.

MicroRNAs (miRNAs) have emerged as important cellular regulators (tumor suppressors, pro-oncogenic factors) of cancer and metastasis. Most published ...