We thank Wei Sheng Tan, Luanne Chiang Xue Yen, Xinyi Lee, and Yingying Chung for providing technical assistance for the preparation of the day 32 hESC-derived photoreceptor progenitors after cryopreservation. This work was supported in part by grants from the National Medical Research Council Young Investigator Research Grant Award (NMRC/OFYIRG/0042/2017) and National Research Foundation 24th Competitive Research Program Grant (CRP24-2020-0083) to H.G.T.

The in vivo experiments were done in accordance with the guidelines and protocol approved by the Institutional Animal Care and Use Committee of SingHealth (IACUC) and the Association for Research in Vision and Ophthalmology (ARVO) Statement for the use of animals in Ophthalmic and Vision Research. The pups were immunosuppressed from P17 (pre-transplantation) to P30 (post-transplantation) by feeding them drinking water containing cyclosporine (260 g/L).
1. Preparation of Day 32 hESC-derived photoreceptor progenitors after cryopreservation
<ol>
	<li>Pre-warm photoreceptor differentiation medium (PRDM) in a 37 &#176;C water bath.</li>
	<li>Retrieve a cryovial containing day 32 hESC-derived photoreceptor progenitor cells from liquid nitrogen. Keep on dry ice.</li>
	<li>Thaw the cryovial at 37 &#176;C in a water bath for 3-5 min. Resuspend the day 32 cells in 1 mL of PRDM and centrifuge at 130 x g for 4 min.</li>
	<li>Remove the supernatant and resuspend cells in 1 mL of PRDM.</li>
	<li>Remove 10 &#181;L of the mixture for cell counting. Mix cells using 0.2% trypan blue according to the manufacturer&#39;s instructions. Pipette cell mixture into the cell counting chamber slide. Determine cell number and viability by automated cell counter.</li>
	<li>Proceed to the next step when the cell viability is above 70%. Centrifuge the remaining cell suspension at 130 x g for 4 min. Remove the supernatant and resuspend the cell pellet in PRDM at the concentration of 3 x 105 cells/&#181;L for transplantation.</li>
	<li>Observe for visible cell clumps. Resuspend cell clumps repeatedly using a 10 &#181;L pipette tip in the microfuge tube until no visible cell clump is observed. Load into the 33G injection syringe to observe for extrusion of cell solution through the needle.</li>
</ol>
2. Sub-retinal delivery of the hESCs in rd10 mice
<ol>
	<li>Preparation of the animals
	<ol>
		<li>Anaesthetize the mouse (P20, male/female, 3-6 g) using a combination of ketamine (20 mg/kg body weight) and xylazine (2 mg/kg body weight) in a 1 mL tuberculin syringe attached to a 27G needle by intraperitoneal approach. Administer a subcutaneous injection of buprenorphine (0.05 mg/kg) to the mouse as a pre-emptive analgesic.</li>
		<li>After administering the anesthesia, instill a drop each of 1% tropicamide and 2.5% phenylephrine for pupil dilation. Apply an ophthalmic gel to the cornea to prevent the eye from dryness and anesthesia-related cataract.</li>
		<li>Place the mouse in an empty cage until fully anesthetized. Assess the proper anesthetic level by pinching the paw pad and confirm if the animal does not react to the hard pinch.</li>
		<li>When the mouse is fully anesthetized, place the animal on a warm pad set at 38 &#176;C.</li>
	</ol>
	</li>
	<li>Sub-retinal delivery of the cells
	<ol>
		<li>To use a 1-port trans-vitreal pars plana approach, as done here, perform sub-retinal injection in a sterile environment. Use an upright operating microscope with a direct light path to perform the injection.</li>
		<li>Prepare the 10 &#181;L glass syringe by removing the needle hub. Mount the 33G blunt needle onto the glass syringe. Take the metal hub cover and carefully secure the needle on the syringe.</li>
		<li>Flush with distilled water to check for any signs of leakage and patency of the needle. Empty the syringe and put it at the side carefully.</li>
		<li>Place the anesthetized mouse on a pillow, with the treatment eye looking up straight to the objective of the microscope. Apply the 0.5% proparacaine hydrochloride and wait for 30 s. Apply 150 &#181;L of ophthalmic gel on the eye and place a round cover slip on it.</li>
		<li>Perform a rough examination of the eye by observing the cornea, iris, pupil, lens, and conjunctiva. Through the pupil, visualize the fundus of the mouse eye by adjusting the focal plane. Adjust the head until the optic head is positioned at the center of the pupil and minimize the movement of the head by proper positioning on the pillow.</li>
		<li>Gently tap the base of the tube with the hESCs multiple times to get a uniform cell suspension. By using the 10 &#181;L glass microliter syringe with a 33G blunt needle, withdraw 2 &#181;L of cells/media. Withdraw the cells right before the injection to avoid cell settlement/clumping in the syringe.</li>
		<li>Using a 30G disposable needle, make a sclerectomy wound 2 mm behind the limbus. Keep the angle of the needle at ~45&#176; to avoid touching the lens. Once the tip of the needle is visualized in the eye, gently withdraw the needle. Discard the needle into the sharp bin after use to avoid needle prick injury.</li>
		<li>Take the glass syringe and insert the blunt needle into the sclerectomy wound. Without touching the lens, advance the blunt needle until it reaches the opposite retina of the entry wound. Ensure the injection area is clear of major retinal blood vessels to avoid bleeding.</li>
		<li>Gently penetrate the retina until a pressure branch on the sclera is seen. Keep the blunt end of the needle parallel to the sclera to avoid leaking of the cells into the vitreous space.</li>
		<li>Slowly inject 2 &#181;L of cell suspension or PRDM media (control) into the sub-retinal space while gentle pressure is maintained on the syringe. With a successful injection, a visible bleb (i.e., raised retina with the cell suspension/media in it) should be formed at the injection site. 
		NOTE: Only gentle pressure should be used during injection to avoid retina tearing and blockage of the cells at the needle tip.</li>
		<li>After confirming the bleb, wait for 10 s to let the cells settle down. Gently retract the needle from the eye.</li>
	</ol>
	</li>
	<li>Intraoperative optical coherence tomography (OCT) scanning of the bleb (optional)
	<ol>
		<li>Position the mouse eye to visualize the bleb under the microscope by slowly moving the head. Secure the position by gently holding the head. No additional pupil dilation is necessary. Do not remove the cover slip and the gel on the eye. It gives a clear optical media to visualize the bleb.</li>
		<li>Perform the intra-operative OCT using the built-in iOCT function of the operating microscope.</li>
		<li>Press the Cube option on the OCT screen and position the scanning area on the bleb by pressing the Arrow buttons. Adjust the OCT by sliding Centering and Focus for the best OCT quality. Press Capture/Scan to acquire the OCT scan of the bleb area. Review the images to check the quality of the scans.</li>
	</ol>
	</li>
	<li>Recovery
	<ol>
		<li>Remove the cover slip and clean the gel from the eye with gauze. Apply antibiotic ointment one time after the injection to prevent infection.</li>
		<li>Allow the animal to recover from anesthesia under a warm light until they regain sufficient consciousness to maintain sternal recumbency and return to the home cage. Monitor the animal for at least 3 days post-injection for signs of inflammation, infection, and distress. 
		NOTE: The 150 W warm light should be at least 30 cm away from the animal cage, and caution should be taken to avoid a burn injury.</li>
		<li>Administer a subcutaneous injection of buprenorphine (0.05 mg/kg) 8 hourly for 1 day or per veterinarian&#8217;s recommendation. If inflammation or infection of the eye is observed, consult a veterinary professional for appropriate treatment.</li>
	</ol>
	</li>
	<li>Cleaning and sterilization of the instruments
	<ol>
		<li>Flush the 10 &#181;L glass microliter syringe and the 33G blunt needle with 100% ethanol 10x. Wash away the ethanol by flashing the syringe with distilled water.</li>
		<li>Dissemble the glass syringe and the needle. Dry the syringe for storage.</li>
	</ol>
	</li>
</ol>

Revitalizing Sight: Stem Cell Therapy for Retinal Degeneration

Hwee Goon Tay is a co-founder of Alder Therapeutics AB. Other authors declare no competing interests.

The sub-retinal injection has been used for cell suspension transplantation to treat RPE and retinal diseases23,25,26,27,28,31,40. This approach is highly essential in rodent studies not only for cell transplantation and gene therapy approaches but also to evaluate novel therapeutic compound...

Inherited retinal diseases and age-related macular degeneration lead to photoreceptor cell loss and eventual blindness. The retinal photoreceptor is the outer segment layer of the retina comprised of specialized cells responsible for phototransduction (i.e., conversion of light to neuronal signals). The rod and cone photoreceptor cells are adjacent to the retinal pigmented layer (RPE)1. Photoreceptor cell replacement therapy to compensate the cell loss has been an emerging and developing therapeutic approach. Embryonic stem cells (ESCs)2,3,4, induced pluripotent stem cells (iPSCs)-derived RPE cells, and retinal progenitor cells (RPCs)4,5,6,7,8 were used to restore the damaged photoreceptor cells. Sub-retinal space, a confined space between the retina and the RPE, is an attractive location to deposit these cells to replace damaged photoreceptor cells, RPE, and Mueller cells due to its vicinity9,10,11.
Gene and cell therapies have been utilizing the sub-retinal space for regenerative medicine for various retinal diseases in pre-clinical studies. This includes the delivery of functional copies of the gene or gene editing tools in the form of either anti-sense oligonucleotide therapy12,13 or CRISPR/Cas9 or base editing via adeno-associated virus (AAV) based strategy14,15,16, implantation of materials (e.g., RPE sheet, retinal prosthetics17,18,19) and differentiated stem cell-derived retinal organoids20,21,22 to treat retinal and RPE-related diseases. Clinical trials using hESC-RPE31 in the sub-retinal space to treat RPE65-associated Leber congenital amaurosis (LCA)23,24, CNGA3-linked achromatopsia25, MERTK-associated retinitis pigmentosa26, choroideremia27,28,29,30 have been proven to be an effective approach. Direct injection of cells to the vicinity of the damaged area greatly improves the chance of cell settlement at the appropriate region, synaptic integration, and eventual visual improvement.
Even though sub-retinal injection in human and large-eyed models (i.e., pig32,33,34,35, rabbit36,37,38,39,40, and non-human primate41,42,43) has been&#160;established, such injection in the murine model is still challenging due to the constrain of the eyeball size and enormous lens occupying the mouse eye44,45,46. However, genetically modified models are only readily available in small animals and not in large animals (i.e., rabbits and non-human primates), therefore sub-retinal injection in mice draws attention to investigate novel therapeutic approaches in retinal genetic disorders. Three major approaches are being used to deliver cells or AAVs into the sub-retinal space, namely the trans-corneal route, trans-scleral route, and the pars plana route (See Figure 2). Trans-corneal and trans-scleral routes are associated with cataract formation, synechiae, choroidal bleeding, and reflux from the injection site11,44,45,47,48,49. We adopted the pars plana approach as a direct visualization of the injection process, and the injection site can be achieved in real-time under the microscope.
We recently described a method that can differentiate human embryonic stem cells (hESCs) into photoreceptor progenitors under xenofree, chemically defined conditions using recombinant human retina-specific laminin isoform LN523. Since LN523 was found to be present in the retina, we hypothesized that the extracellular matrix niche of the human retina could be recapitulated in vitro and thereby support photoreceptor differentiation from the hESCs36. Single-cell transcriptomic analysis showed that photoreceptor progenitors co-expressing cone-rod homeobox and recoverin were generated after 32 days. A retinal degeneration 10 (rd10) mutant mouse model that mimics autosomal human retinitis pigmentosa was used to evaluate the efficacy of the day 32 hESC-derived photoreceptor progenitors in-vivo. The hESC-derived photoreceptor progenitor cells were injected into the sub-retinal space of rd10 mice at P20, where photoceptor dysfunction and degeneration are ongoing36. Here, we describe a detailed protocol for the preparation of the post-cryopreserved hESC-derived photoreceptor progenitors and delivery into the sub-retinal space of rd10 mice. This method can also be used to administer AAVs, cell suspensions, peptides, or chemicals into the sub-retinal space in mice.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.3% Tobramycin</td>
			<td>Novartis</td>
			<td>NDC &#160;0078-0813-01</td>
			<td>Tobrex (3.5 g)</td>
		</tr>
		<tr>
			<td>0.3% Tobramycin and 0.1% Dexamethasone</td>
			<td>Novartis</td>
			<td>NDC 0078-0876-01</td>
			<td>Tobradex (3.5 g)</td>
		</tr>
		<tr>
			<td>0.5% Proparacaine hydrochloride</td>
			<td>Alcon</td>
			<td>NDC 0998-0016-15</td>
			<td>0.5% Alcaine (15 mL)</td>
		</tr>
		<tr>
			<td>1 mL Tuberculin syringe</td>
			<td>Turemo</td>
			<td>SS01T2713</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Tropicamide</td>
			<td>Alcon</td>
			<td>NDC 0998-0355-15</td>
			<td>1% Mydriacyl (15 mL)</td>
		</tr>
		<tr>
			<td>2.5% Phenylephrine hydrochloride</td>
			<td>Alcon</td>
			<td>NDC 0998-0342-05</td>
			<td>2.5% Mydfrin (5 mL)</td>
		</tr>
		<tr>
			<td>24-well tissue culture plate</td>
			<td>Costar</td>
			<td>3526</td>
			<td></td>
		</tr>
		<tr>
			<td>30 G Disposable needle</td>
			<td>Becton Dickinson (BD)</td>
			<td>305128</td>
			<td></td>
		</tr>
		<tr>
			<td>33 G, 20 mm length blunt needles</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated Cell Counter</td>
			<td>NanoEnTek</td>
			<td>Model: Eve</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 without Vitamin A</td>
			<td>Life Technologies</td>
			<td>12587001</td>
			<td>2%36</td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Ceva</td>
			<td></td>
			<td>Vetergesic vet (0.3 mg/mL)</td>
		</tr>
		<tr>
			<td>CKI-7</td>
			<td>Sigma</td>
			<td>C0742</td>
			<td>5 &#181;M36</td>
		</tr>
		<tr>
			<td>Cyclosporine</td>
			<td>Novartis</td>
			<td></td>
			<td>260 g/L in drinking water</td>
		</tr>
		<tr>
			<td>Day 32 hESC-derived photoreceptor progenitor cells</td>
			<td>DUKE-NUS Medical School</td>
			<td></td>
			<td>Human embryonic stem cells are differentiated for 32 days. See protocol in Ref 36.</td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Winner Industries Co. Ltd.</td>
			<td>1SNW475-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glasgow Minimum Essential Medium</td>
			<td>Gibco</td>
			<td>11710&#8211;035</td>
			<td></td>
		</tr>
		<tr>
			<td>hESC cell line H1</td>
			<td>WiCell Research Institute</td>
			<td>WA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Human brain-derived neurotrophic factor (BDNF)</td>
			<td>Peprotech</td>
			<td>450-02-50</td>
			<td>10 ng/mL36</td>
		</tr>
		<tr>
			<td>Human ciliary neurotrophic factor (CNTF)</td>
			<td>Prospec-Tany Technogene</td>
			<td>CYT-272</td>
			<td>10 ng/mL36</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride (100 mg/mL)</td>
			<td>Ceva Sant&#233; Animale</td>
			<td>KETALAB03</td>
			<td></td>
		</tr>
		<tr>
			<td>LN-521</td>
			<td>Biolamina</td>
			<td>LN521-02</td>
			<td>1 &#181;g36</td>
		</tr>
		<tr>
			<td>mFreSR</td>
			<td>STEMCELL Technologies</td>
			<td>5854</td>
			<td></td>
		</tr>
		<tr>
			<td>Microlitre glass syringe (10 mL)</td>
			<td>Hamilton</td>
			<td>7653-01</td>
			<td></td>
		</tr>
		<tr>
			<td>N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT)</td>
			<td>Selleckchem</td>
			<td>S2215</td>
			<td>10 &#181;M36</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Life Technologies</td>
			<td>A13707-01</td>
			<td>1%36</td>
		</tr>
		<tr>
			<td>Non-essential amino acids (NEAA)</td>
			<td>Gibco</td>
			<td>11140&#8211;050</td>
			<td>1x36</td>
		</tr>
		<tr>
			<td>NutriStem XF Media</td>
			<td>Satorius</td>
			<td>05-100-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Operating microscope</td>
			<td>Zeiss</td>
			<td>OPMI LUMERA 700</td>
			<td>With Built-in iOCT function</td>
		</tr>
		<tr>
			<td>PRDM (Photoreceptor differentiation medium, 50ml)</td>
			<td>DUKE-NUS Medical School</td>
			<td></td>
			<td>See media composition36. Basal Medium, 10 &#181;M DAPT, 10 ng/mL BDNF, 10 ng/mL CNTF, 0.5 &#181;M Retinoic acid, 2% B27 and 1% N2. Basal Medium: 1x GMEM, 1 mM sodium pyruvate, 0.1 mM B-mercaptoethanol, 1x Non-essential amino acids (NEAA).</td>
		</tr>
		<tr>
			<td>Pyruvate</td>
			<td>Gibco</td>
			<td>11360&#8211;070</td>
			<td>1 mM36</td>
		</tr>
		<tr>
			<td>Rd10 mice</td>
			<td>Jackson Laboratory</td>
			<td>B6.CXB1-Pde6brd10/J mice</td>
			<td>Gender: male/female, Age: P20 (injection), Weight: 3-6 g&#160;</td>
		</tr>
		<tr>
			<td>Retinoic acid</td>
			<td>Tocris Bioscience</td>
			<td>0695/50</td>
			<td>0.5 &#181;M36</td>
		</tr>
		<tr>
			<td>Round Cover Slip (12 mm)</td>
			<td>Fisher Scientific</td>
			<td>12-545-80</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Sigma</td>
			<td>S4317</td>
			<td>0.5 &#181;M36</td>
		</tr>
		<tr>
			<td>Vidisic Gel (10 g)</td>
			<td>Dr. Gerhard Mann</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine hydrochloride (20 mg/mL)</td>
			<td>Troy Laboratories</td>
			<td>LI0605</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Life Technologies</td>
			<td>21985&#8211;023</td>
			<td>0.1 mM36</td>
		</tr>
	</tbody>
</table>

sub-retinal delivery of human embryonic stem cell derived photoreceptor progenitors in rd10 mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal diseases at advanced stages. We have shown human recombinant retina-specific laminin isoform matrix is able to support the differentiation of human embryonic stem cells (hESCs) to photoreceptor progenitors. In addition, sub-retinal injection of these cells has also shown partial restoration in the rd10 rodent and rabbit models. Sub-retinal injection is known to be an established method that has been used to deliver pharmaceutical compounds to the photoreceptor cells and retinal pigmented epithelial (RPE) layer of the eye due to its proximity to the target space. It has also been used to deliver adeno-associated viral vectors into the sub-retinal space to treat retinal diseases. The sub-retinal delivery of pharmaceutical compounds and cells in the murine model is challenging due to the constraint in the size of the murine eyeball. This protocol describes the detailed procedure for the preparation of hESC-derived photoreceptor progenitor cells for injection and the sub-retinal delivery technique of these cells in genetic retinitis pigmentosa mutant, rd10 mice. This approach allows cell therapy to the targeted area, in particular the outer nuclear layer of the retina, where diseases leading to photoreceptor degeneration occur.

The 10 &#181;L glass syringe was assembled according to the manufacturer&#39;s instructions (Figure 1), and the blunt needle used to deliver the cell suspension/media is shown in Figure 1B. Different approaches for sub-retinal injection are illustrated in Figure 2. We describe the pars plana approach in this protocol (Figure 2C). The blunt needle mounted on a glass syringe was inserted through a sclerot...

Watch this Scientific Journal Video about Advancing Vision Restoration â€” Stem Cell-Based Therapy for Retinal Diseases at JoVE.com

Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Sub-Retinal Delivery of Human Embryonic Stem Cell Derived Photoreceptor Progenitors in rd10 Mice

Regeneration of photoreceptor cells using human pluripotent stem cells is a promising therapy for the treatment of both hereditary and aging retinal ...

sub-retinal-delivery-human-embryonic-stem-cell-derived-photoreceptor

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802 Views.  Singapore Eye Research Institute. We describe a detailed protocol for the preparation of post-cryopreserved hESC-derived photoreceptor progenitor cells and the sub-retinal delivery of these cells in rd10 mice.

Video: Author Spotlight: Advancing Vision Restoration - Stem Cell-Based Therapy for Retinal Diseases 

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and finding new sources for implantable cells are the focus of recent research4,5. Investigating the effectiveness of cell therapies is not an easy task and new tools are needed to investigate the mechanisms involved in the treatment process6. We designed an experimental protocol of ischemia/reperfusion in order to allow the observation of cellular connections and even subcellular mechanisms during ischemia/reperfusion injury and after stem cell transplantation and to evaluate the efficacy of cell therapy. H9c2 cardiomyoblast cells were placed onto cell culture plates7,8. Ischemia was simulated with 150 minutes in a glucose free medium with oxygen level below 0.5%. Then, normal media and oxygen levels were reintroduced to simulate reperfusion. After oxygen glucose deprivation, the damaged cells were treated with transplantation of labeled human bone marrow derived mesenchymal stem cells by adding them to the culture. Mesenchymal stem cells are preferred in clinical trials because they are easily accessible with minimal invasive surgery, easily expandable and autologous. After 24 hours of co-cultivation, cells were stained with calcein and ethidium-homodimer to differentiate between live and dead cells. This setup allowed us to investigate the intercellular connections using confocal fluorescent microscopy and to quantify the survival rate of postischemic cells by flow cytometry. Confocal microscopy showed the interactions of the two cell populations such as cell fusion and formation of intercellular nanotubes. Flow cytometry analysis revealed 3 clusters of damaged cells which can be plotted on a graph and analyzed statistically. These populations can be investigated separately and conclusions can be drawn on these data on the effectiveness of the simulated therapeutical approach.

Stem Cell Transplantation in an in vitro Simulated Ischemia/Reperfusion Model

We demonstrate how to set up an in vitro ischemia/reperfusion model and how to evaluate the effect of stem cell therapy on postischemic cardiac cells.

Stem cell transplantation protocols are finding their way into clinical practice1,2,3. Getting better results, making the protocols more robust, and ...

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Parabiosis in Mice: A Detailed Protocol

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of microvasculature at the site of inflammation. Parabiotic partners share their circulating antigens and thus are free of adverse immune reaction. First described by Paul Bert in 18641, the parabiosis surgery was refined by Bunster and Meyer in 1933 to improve animal survival2. In the current protocol, two mice are surgically joined following a modification of the Bunster and Meyer technique. Animals are connected through the elbow and knee joints followed by attachment of the skin allowing firm support that prevents strain on the sutured skin. Herein, we describe in detail the parabiotic joining of a ubiquitous GFP expressing mouse to a wild type (WT) mouse. Two weeks after the procedure, the pair is separated and GFP positive cells can be detected by flow cytometric analysis in the blood circulation of the WT mouse. The blood chimerism allows one to examine the contribution of the circulating cells from one animal in the other.

Parabiotic joining of two organisms leads to the development of a shared circulatory system. In this protocol, we describe the surgical steps to form a parabiotic connection between a wild-type mouse and a constitutive GFP-expressing mouse.

Parabiosis is a surgical union of two organisms allowing sharing of the blood circulation. Attaching the skin of two animals promotes formation of ...

Ultrasound-guided Transthoracic Intramyocardial Injection in Mice

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. Experiments involving myocardial injection are currently performed by direct surgical access through a thoracotomy. While convenient when performed at the time of another experimental manipulation such as coronary artery ligation, the need for an invasive procedure for intramyocardial delivery limits potential experimental designs. With ever improving ultrasound resolution and advanced noninvasive imaging modalities, it is now feasible to routinely perform ultrasound-guided, percutaneous intramyocardial injection. This modality efficiently and reliably delivers agents to a targeted region of myocardium. Advantages of this technique include the avoidance of surgical morbidity, the facility to target regions of myocardium selectively under ultrasound guidance, and the opportunity to deliver injectate to the myocardium at multiple, predetermined time intervals. With practiced technique, complications from intramyocardial injection are rare, and mice quickly return to normal activity on recovery from anesthetic. Following the steps outlined in this protocol, the operator with basic echocardiography experience can quickly become competent in this versatile, minimally invasive technique.

Echocardiography-guided percutaneous intramyocardial injection represents an efficient, reliable, and targetable modality for the delivery of gene transfer agents or cells into the murine heart. Following the steps outlined in this protocol, the operator can quickly become competent in this versatile, minimally invasive technique.

Murine models of cardiovascular disease are important for investigating pathophysiological mechanisms and exploring potential regenerative therapies. ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Expression of Exogenous Cytokine in Patient-derived Xenografts via Injection with a Cytokine-transduced Stromal Cell Line

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying the mechanisms of normal and malignant hematopoiesis and are the gold standard for identifying effective chemotherapies for many malignancies. PDX models are possible because many of the mouse cytokines also act on human cells. However, this is not the case for all cytokines, including many that are critical for studying normal and malignant hematopoiesis in human cells. Techniques that engineer mice to produce human cytokines (transgenic and knock-in models) require significant expense before the usefulness of the model has been demonstrated. Other techniques are labor intensive (injection of recombinant cytokine or lentivirus) and in some cases require high levels of technical expertise (hydrodynamic injection of DNA). This report describes a simple method for generating PDX mice that have exogenous human cytokine (TSLP, thymic stromal lymphopoietin) via weekly intraperitoneal injection of stroma that have been transduced to overexpress this cytokine. Use of this method provides an in vivo source of continuous cytokine production that achieves physiological levels of circulating human cytokine in the mouse. Plasma levels of human cytokine can be varied based on the number of stromal cells injected, and cytokine production can be initiated at any point in the experiment. This method also includes cytokine-negative control mice that are similarly produced, but through intraperitoneal injection of stroma transduced with a control vector. We have previously demonstrated that leukemia cells harvested from TSLP-expressing PDX, as compared to control PDX, exhibit a gene expression pattern more like the original patient sample. Together the cytokine-producing and cytokine-negative PDX mice produced by this method provide a model system that we have used successfully to study the role of TSLP in normal and malignant hematopoiesis.

Described here is a method for producing exogenous cytokine in patient-derived xenograft (PDX) mice via weekly intraperitoneal injection of a cytokine-transduced stromal cell line. This method broadens the utility of PDX and provides the option for transient or sustained exogenous cytokine delivery in a multitude of PDX models.

Patient-derived xenograft (PDX) mice are produced by transplanting human cells into immune deficient mice. These models are an important tool for studying ...

Intratracheal Administration of Dry Powder Formulation in Mice

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper introduces a noninvasive method of intratracheal delivery of dry powder formulation in mice. A dry powder loading device that consists of a 200 &#181;L gel loading pipette tip connected to an 1 mL syringe via a three-way stopcock is presented. A small amount of dry powder (1-2 mg) is loaded into the pipette tip and dispersed by 0.6 mL of air in the syringe. Because pipette tips are disposable and inexpensive, different dry powder formulations can be loaded into different tips in advance. Various formulations can be evaluated in the same animal experiment without device cleaning and dose refilling, thereby saving time and eliminating the risk of cross-contamination from residual powder. The extent of powder dispersion can be inspected by the amount of powder remaining in the pipette tip. A protocol of intubation in mouse with a custom-made light source and a guiding cannula is included. Proper intubation is one of the key factors that influences the intratracheal delivery of dry powder formulation to the deep lung region of the mouse.

Dry powder formulations for inhalation have great potential in treating respiratory diseases. Before entering human studies, it is necessary to evaluate the efficacy of the dry powder formulation in preclinical studies. A simple and noninvasive method of the administration of dry powder in mice through the intratracheal route is presented.

In the development of inhalable dry powder formulations, it is essential to evaluate their biological activities in preclinical animal models. This paper ...

A Preclinical Model of Exertional Heat Stroke in Mice

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, whereas exertional heat stroke (EHS) occurs during physical activity. EHS differs from CHS in etiology, clinical presentation, and sequelae of multi-organ dysfunction. Until recently, only models of CHS have been well established. This protocol aims to provide guidelines for a refined preclinical mouse model of EHS that is free from major limiting factors such as the use of anesthesia, restraint, rectal probes, or electric shock. Male and female C57Bl/6 mice, instrumented with core temperature (Tc) telemetric probes were utilized in this model. For familiarization with the running mode, mice undergo 3 weeks of training using both voluntary and forced running wheels. Thereafter, mice run on a forced wheel inside a climatic chamber set at 37.5 &#176;C and 40%-50% relative humidity (RH) until displaying symptom limitation (e.g., loss of consciousness) at Tc of 42.1-42.5 &#176;C, although suitable results can be obtained at chamber temperatures between 34.5-39.5 &#176;C and humidity between 30%-90%. Depending on the desired severity, mice are removed from the chamber immediately for recovery in ambient temperature or remain in the heated chamber for a longer duration, inducing a more severe exposure and a higher incidence of mortality. Results are compared with sham-matched exercise controls (EXC) and/or na&#239;ve controls (NC). The model mirrors many of the pathophysiological outcomes observed in human EHS, including loss of consciousness, severe hyperthermia, multi-organ damage as well as inflammatory cytokine release, and acute phase responses of the immune system. This model is ideal for hypothesis-driven research to test preventative and therapeutic strategies that may delay the onset of EHS or reduce the multi-organ damage that characterizes this manifestation.

The protocol describes the development of a standardized, repeatable, preclinical model of exertional heat stroke (EHS) in mice free from adverse external stimuli such as electric shock. The model provides a platform for mechanistic, preventative, and therapeutic studies.

Heat stroke is the most severe manifestation of heat-related illnesses. Classic heat stroke (CHS), also known as passive heat stroke, occurs at rest, ...