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)

<ol>
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E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentivector-mediated+clonal+tracking+reveals+intrinsic+heterogeneity+in+the+human+hematopoietic+stem+cell+compartment+and+culture-induced+stem+cell+impairment.">Lentivector-mediated clonal tracking reveals intrinsic heterogeneity in the human hematopoietic stem cell compartment and culture-induced stem cell impairment.</a> Blood. 103 (2), 545-552 (2004).</li><li>Piras, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+vectors+escape+innate+sensing+but+trigger+p53+in+human+hematopoietic+stem+and+progenitor+cells.">Lentiviral vectors escape innate sensing but trigger p53 in human hematopoietic stem and progenitor cells.</a> EMBO Molecular Medicine. 9 (9), 1198-1211 (2017).</li><li>Christopher, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+expansion+of+human+CD34+CD133+CD90++hematopoietic+stem+cells+enhances+gene-modified+cell+frequency+for+gene+therapy.">Preferential expansion of human CD34+CD133+CD90+ hematopoietic stem cells enhances gene-modified cell frequency for gene therapy.</a> Human Gene Therapy. 33 (3-4), 188-201 (2021).</li><li>Karuppusamy, K. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CCR5+gene+edited+CD34++CD90++hematopoietic+stem+cell+population+serves+as+an+optimal+graft+source+for+HIV+gene+therapy.">The CCR5 gene edited CD34+ CD90+ hematopoietic stem cell population serves as an optimal graft source for HIV gene therapy.</a> Frontiers in Immunology. 13, 792684 (2022).</li><li>Hopman, R. K., DiPersio, J. 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The authors declare that no competing financial interests exist.

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>
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			<td >Mouse Pie Cage</td>
			<td>FISCHER SCIENTIFIC</td>
			<td>50-195-5140</td>
			<td></td>
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			<td >polystyrene round-bottom tube (12 x 75 mm)</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >38007</td>
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			<td >Primer3</td>
			<td >Whitehead Institute for Biomedical Research</td>
			<td >https://primer3.ut.ee/</td>
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			<td >QuickExtract&#8482; DNA Extraction Solution</td>
			<td >Lucigen</td>
			<td >QE09050</td>
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			<td >Reserveratrol</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >72862</td>
			<td></td>
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			<td >SCF</td>
			<td >PEPROTECH</td>
			<td >300-07-1000</td>
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			<td >SFEM-II</td>
			<td >STEMCELL TECHNOLOGIES</td>
			<td >9655</td>
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			<td >sgRNA</td>
			<td >SYNTHEGO</td>
			<td >-&#160;</td>
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			<td >STEMCELL TECHNOLOGIES</td>
			<td >72342</td>
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			<td >ICE analysis tool</td>
			<td >SYNTHEGO</td>
			<td >https://ice.synthego.com/</td>
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			<td >Tris-EDTA buffer solution (TE) 1X</td>
			<td >SYNTHEGO</td>
			<td >Supplied with gRNA&#160;</td>
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			<td >Thermocycler</td>
			<td >APPLIED BIOSYSTEMS</td>
			<td >4375305</td>
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			<td >TPO</td>
			<td >PEPROTECH</td>
			<td >300-18-1000</td>
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			<td >TCL046</td>
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			<td >UM171</td>
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			<td >Xylazine</td>
			<td >XYLAXIN - INDIAN IMMUNOLOGICALS LIMITED</td>
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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 ...

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

Research

JoVE Journal

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3.4K Views.  Christian Medical College campus. The present protocol describes an optimized hematopoietic stem and progenitor cell (HSPC) culture procedure for the robust engraftment of gene-edited cells in vivo.

Video: 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

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

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, ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...

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-&#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 ...