This work was supported by a Grant-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science (JSPS) (grant nos. JP19K10259 and 22K10032 to MAI).

This protocol uses 4-week-old male nude mice. House four mice in a cage under a 12 h light/dark cycle at 22&#8722;24 &#176;C and 50%&#8722;70% relative humidity. All animal experiments were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee of Tokyo Medical and Dental University (approval ID: A2019-204C, A2020-116A, and A2021-121A).
1. Preparation of buffers and reagents
<ol>
	<li>Prepare mesenchymal stem cell growth medium (MSC growth medium) by adding the supplement kit of MSC growth medium to MSC basal media and store it at 4 &#176;C.</li>
	<li>Prepare Dulbecco&#39;s Modified Eagle Medium and Ham&#39;s F12 (DMEM/F-12) medium by adding 10% fetal bovine serum (FBS), 50 &#181;g/mL gentamicin, 200 &#181;M L-alanyl-L-glutamine, and 1 ng/mL basic fibroblastic growth factor to the medium and store it at 4 &#176;C.</li>
	<li>Prepare chondrogenic medium by adding 1% ITS-G supplement, 0.12% bovine serum albumin, 50 &#181;g/mL gentamicin, 0.35 mM L-proline, 100 nM dexamethasone, and 10 ng/mL TGF-&#946;3 to DMEM just before use.</li>
	<li>Prepare hypertrophic medium by adding 10 mM &#946;-glycerophosphate, 0.12% bovine serum albumin, 10 nM dexamethasone, 200 &#181;M ascorbate-2-phosphate, 50 nM L-thyroxine, 50 &#181;g/mL gentamycin, and 50 pg/mL IL-1&#946; to DMEM just before use.</li>
	<li>Coat a 24-well culture dish with 200 &#181;L of melted paraffin pre-heated at 60 &#176;C. Allow to stand at room temperature until the paraffin is set.</li>
</ol>
2. Expansion of human MSCs
NOTE: Prior to starting the experiments, MSCs with a high potential for MSC chondrogenesis should be selected in micro mass culture as described in17.
<ol>
	<li>According to the manufacturer&#39;s instructions, culture primary human bone marrow-derived MSCs (passage 2) with MSC growth medium in a 10 cm dish at a density of 5 x 103 cells/cm2 at 37 &#176;C in 5% CO2 and 95% humidified air incubator.</li>
	<li>Change the medium every 2-3 days. Once the cells reach 80%-90% confluency, aspirate the medium from the cell culture dish using a vacuum pump, wash with 5 mL of PBS, and then aspirate the solution with a vacuum pump.</li>
	<li>Add 1 mL of 0.05% trypsin/0.02% EDTA solution to the dish. Incubate the dish for 5-10 min at 37 &#176;C.</li>
	<li>Add 1 mL of MSC growth medium to stop the enzymatic reaction. Transfer the cell suspension to a 50 mL tube.</li>
	<li>Combine the cell suspension from other dishes in the 50 mL tube and keep it on ice.</li>
	<li>Use the cell counter to count the cells by mixing 10 &#181;L of the cell suspension with 10 &#181;L of 0.4% trypan blue stain.</li>
	<li>Centrifuge the remaining cell suspension at 220 x g for 3 min at room temperature. Remove the supernatant and add cryopreservation solution at a concentration of 1.0 x 106 cells per mL.</li>
	<li>Dispense 1 mL of the cell suspension into each cryotube and store at -80 &#176;C for 12 h before transferring to liquid nitrogen. The resulting frozen stocks (passage 3) are used for this protocol.</li>
	<li>For the experiments, seed the stocked MSCs in a 10 cm dish at a density of 5 x 103 cells/cm2. Culture cells until 80%-90% confluent (passage 4) in DMEM/F-12 medium.</li>
</ol>
3. Preparation of MSC-encapsulated hydrogels
<ol>
	<li>Trypsinize the cells and prepare cell suspension as described in steps 2.3 - 2.6.</li>
	<li>To make 10 constructs (2.5 x 105 cells per construct), aliquot the cell suspension containing 2.5 x 106 cells into a 1.5 mL microtube.</li>
	<li>Centrifuge the suspension at 220 x g for 3 min, remove the supernatant with a vacuum pump, and keep it on ice.</li>
	<li>Bring the thiol-modified hyaluronic acid (HA), thiol-reactive crosslinker, polyethylene glycol diacrylate, and degassed water bottles to room temperature.</li>
	<li>Under sterile conditions, add 1.0 mL of degassed water to the HA-containing bottle (see manufacturer&#39;s instructions) using a syringe with a needle.</li>
	<li>Vortex occasionally warming to 37 &#176;C until the solution becomes clear, then place on ice. It takes less than 30 min for the solids to dissolve completely.</li>
	<li>Under sterile conditions, add 0.5 mL of degassed water to the crosslinker bottle using a syringe with a needle. Dissolve by inverting several times, then place on ice.</li>
	<li>Add 120 &#181;L of the dissolved HA solution to the aliquoted cell pellet (2.5 x 106 cells). Resuspend the cell pellet by pipetting back and forth.</li>
	<li>Add 30 &#181;L of the dissolved crosslinker solution to the tube containing the HA and cells (step 3.8) and combine by tapping the tube, and then spin down briefly. Combine the HA solution and the crosslinker solution in a 4:1 ratio.</li>
	<li>Drop 15 &#181;L of the combined solution containing MSCs (2.5 x 105 cells) onto the paraffin-coated 24-well plate and allow it to solidify at 37 &#176;C for 30 min (Figure 2). Due to high viscosity, prepare at least 10% extra amount of the cell/hydrogel mixture than needed. 
	&#8203;NOTE: The characteristics of the hydrogel have been described previously39.</li>
</ol>
4. In vitro differentiation conditions
<ol>
	<li>Add 0.5 mL of chondrogenic differentiation medium to a construct of MSC-seeded HA hydrogels. Change the medium every 2-3 days.</li>
	<li>After 3 weeks, switch to hypertrophic differentiation medium (0.5 mL per well) and culture the constructs for an additional 2 weeks.</li>
</ol>
5. In vivo implantation of MSCs
<ol>
	<li>After a total of 5 weeks of in vitro culture, implant the constructs subcutaneously into the back of nude mice as described previously38,40.</li>
	<li>Weigh mice and administer a combination anesthetic prepared with 0.75 mg/kg body weight (b.w.) medetomidine, 4.0 mg/kg b.w. midazolam, and 5.0 mg/kg b.w. butorphanol by intraperitoneal injection as described38.</li>
	<li>Confirm adequate anesthesia by immobility to surgical stimuli and monitor respiratory depth by slow, regular chest movements and proper oxygen supply by the color of the pink mucosa periodically during the operation. Use veterinary ointment on the eyes to prevent dryness during anesthesia.</li>
	<li>Place the mouse on a sterile surgical drape in a prone position, sterilize the incision site with 50 ppm hypochlorous acid water (pH 5.0; HAW), and place a perforated surgical drape over the mouse. 
	&#8203;NOTE: Sterilize all surgical materials in an autoclave, ethylene oxide gas, or HAW. The surgeons should wash their fingers and wear surgical gowns and gloves.</li>
	<li>Make two 5 mm long skin incisions 2 cm apart in the shoulder and hip areas along the center line of the spine by using scissors.</li>
	<li>Insert a spatula subcutaneously through the incision to create a subcutaneous pocket.</li>
	<li>Insert two to three constructs (~15 &#181;L each, step 5.1) into each pocket with forceps. HA constructs implanted in the same pocket are prone to fusion very frequently. To prevent fusion, insert one HA construct in each pocket.</li>
	<li>Close the incisions with 4-0 braided silk sutures. Inject the mouse with an antagonist to anesthetics prepared with 0.75 mg/kg b.w. atipamezole as described38.</li>
	<li>While the mice recover from anesthesia, place the mice in sterile recovery cages containing sterile floor bedding without food and water bottles and continuously monitor body temperature, pulse, and respiration.</li>
	<li>Do not leave the mice unattended until it has regained sufficient consciousness to maintain sternal recumbency. After full recovery, return the animals to the breeding room with the other animals.</li>
	<li>Monitor the animals every couple of days during the healing period for any possible complications. Euthanize the mice by carbon dioxide inhalation with little suffering at 4- and 8-weeks post-implantation.</li>
	<li>Incise the skin at the grafted sites and remove the implanted constructs with forceps. Fix the constructs in 4% paraformaldehyde for 16 h and then subject them to analysis.</li>
</ol>
6. Statistical analysis
<ol>
	<li>Report quantitative data as mean &#177; standard deviation (SD). Perform statistical analysis using commercial software.</li>
	<li>Use the Student&#39;s t-test or one-way ANOVA followed by Tukey&#39;s multiple comparisons test to determine significant differences between groups. p&#60;0.05 and p&#60;0.01 indicate significant differences between two groups.</li>
</ol>

Artificial Cartilage Implants for Bone Regeneration via Endochondral Ossification

The authors have declared that no competing interests exist.

Using appropriate scaffold materials that promote the transition from hypertrophic cartilage to bone is a promising approach to scale up MSC-based engineered hypertrophic cartilage grafts and treat bone defects of clinically significant size. Here, we show that HA is an excellent scaffold material to support the differentiation of MSC-based hypertrophic cartilage tissue in vitro and to promote endochondral bone formation in vivo38. Furthermore, in vivo, HA constructs were shown to facilitate the fusion of multiple constructs implanted in the same pocket to form a single integrated bone. Because the interaction of MSCs with scaffold materials significantly affects cartilage and hypertrophic chondrogenesis, selecting suitable scaffold material for ECOs is important for generating clinically effective tissue-engineered cartilage grafts. Cells embedded in HA hydrogels uniformly exhibit a rounded morphology characteristic of chondrocytes from the center to the periphery, indicating that hyaluronan provides an environment conducive to uniform cartilage formation throughout the hydrogel21,30. Unlike HA, collagen interacts with MSCs via integrin binding sequences (RGDs); however, the persistence of this interaction prevents MSCs from further differentiating in the chondrogenic pathway43, which may account for the non-typical chondrocyte cell morphology that is prominent in the periphery of collagen constructs.
If multiple grafts unite to form a single bone, this ECO-mediated approach can be extended to more clinically relevant bone defects. When multiple MSC-based and scaffold-free hypertrophic cartilage were applied to a single bone defect, it was reported that the cartilage grafts integrated with the bone defect. However, adjacent grafts did not integrate with each other22. The approach presented here may offer one possible solution to overcome this limitation. Compared to collagen constructs, multiple grafts of HA constructs had a greater tendency to fuse to form a single bone. Furthermore, bone marrow was formed between fused HA constructs, which may be related to the fact that HA supports uniform cartilage differentiation throughout the hydrogel because hypertrophic cartilage secretes VEGF and provides an environment for the hematopoietic stem-cell niche in bone marrow development 44.
The key to a successful experiment using this protocol is the quality of MSCs. Confirming that the MSCs to be used have sufficiently high chondrogenic differentiation potential in advance is recommended. Second, the size of each construct should not be too large. In our experience, the volume of a single construct is limited to 10-15 &#181;L. Larger constructs may result in insufficient oxygen and nutrient penetration into the constructs, leading to necrosis and poor differentiation in the center of the constructs. Therefore, gel thickness needs to be reduced to improve oxygen and nutrient penetration. In order to reduce gel thickness, it may be helpful to create thin gels on the porous membrane of Transwell inserts14,45. Alternatively, because HA constructs tend to fuse and form integrated bone, it may be adequate to implant many smaller constructs rather than a single large construct, as discussed below.
Limitations of this study include the need to accelerate the remodeling rate of the implanted tissues&#160;to facilitate bone formation.The implanted engineered tissue remained primarily in a hypertrophic calcified cartilage state even 8 weeks post-implantation. In bone formation via the ECO pathway, host-derived cells play a critical role in promoting ECO16,46. Providing additional channels in hypertrophic cartilage grafts has been reported to promote vascular invasion and graft mineralization in vivo20. Such channels serve as conduits for the infiltration of host cells, including osteoblasts, osteoclasts, and hematopoietic precursors, to form new tissue within the construct. Alternatively, it has been reported that multiple small fibrin-encapsulated pellets (&#181;-pellets) composed of chondrogenic differentiated MSCs can be implanted to form integrated bone47. Processing HA constructs into &#181;-pellets could increase the surface area surrounded by TRAP-positive osteoclasts, thus promoting the remodeling rate of cartilage tissue&#160;and enhancing osteogenesis (Figure 6)21,48. Furthermore, implantation of &#181;-pellets would increase the infiltration space of host cells, thereby facilitating angiogenesis and bone marrow development and generating integral bone formation with abundant vascularization. Thus, considering the tendency of HA constructs to fuse and create integrated bone, this &#181;-constructed bone regeneration strategy may accelerate the ECO process of hypertrophic cartilage grafts using HA hydrogels.
In conclusion, HA hydrogels are effective in scaling up tissue-engineered grafts with fewer cells and gels than collagen hydrogels. In addition, HA hydrogels have excellent fusion sensitivity and produce integrated bone with vascularization and marrow development between fused grafts. Therefore, modification of HA constructs to promote host cell infiltration and graft remodeling may lead to the development of implantable materials that can further promote vascularization and bone marrow development. With further optimization, this approach could be a promising alternative to current bone therapies in maxillofacial and orthopedic surgery.

Autologous bone is still the gold standard for repairing bone defects due to trauma, congenital defects, and surgical resection. However, autogenous bone grafting has significant limitations, including donor pain, risk of infection, and limited bone volume that can be isolated from the patients1,2,3,4. Numerous biomaterials have been developed as bone substitutes, combining natural or synthetic polymers with mineralized materials such as calcium phosphate or hydroxyapatite5,6. Bone formation in these engineered materials is usually achieved using the mineralized material as a priming material to allow stem cells to differentiate directly into osteoblasts through the intramembrane ossification (IMO) process7. This process lacks the angiogenic step, resulting in insufficient in vivo vascularization of the graft after implantation8,9,10, and therefore, approaches using such a process may not be optimal for treating large bone defects11.
Strategies applied to recapitulate the endochondral ossification (ECO) process, an innate mechanism in skeletogenesis during development, have been shown to overcome significant problems associated with traditional IMO-based approaches. In ECO, chondrocytes in the cartilage template release vascular endothelial growth factor (VEGF), which promotes vascular infiltration and remodeling of the cartilage template into bone12. The ECO-mediated approach to osteogenesis via cartilage remodeling and angiogenesis, which is also activated during fracture repair, uses artificially created cartilage tissue derived from MSCs as a priming material. Chondrocytes can tolerate hypoxia in bone defects, induce angiogenesis, and convert a vascular-free cartilage graft into angiogenic tissue. Numerous studies have reported that MSC-based cartilage grafts generate bone in vivo by implementing such an ECO program13,14,15,16,17,18,19,20,21.
An essential requirement for the clinical application of this ECO-mediated approach is how to prepare the desired amount of cartilage graft in a clinical setting. Preparing clinical cartilage of a size that fits the actual bone defect is not practical. Therefore, graft cartilage must form bone integrally when multiple fragments are implanted22. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification. Many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo23,24,25,26,27,28,29,30,31,32; however, the optimal support material to meet the clinical application requirements has remained undetermined. Hyaluronic acid (HA) is a biodegradable and biocompatible polysaccharide present in the extracellular matrix of cartilage33. HA interacts with MSCs via surface receptors such as CD44 to support chondrogenic differentiation25,26,28,30,31,32,34. In addition, HA scaffolds promote IMO-mediated osteogenic differentiation of human dental pulp stem cells35, and scaffolds combined with collagen promote ECO-mediated osteogenesis36,37.
Here, we present a method for preparing HA hydrogels using bone marrow-derived adult human MSCs and their use for hypertrophic chondrogenesis in vitro and subsequent endochondral ossification in vivo38. We compared the characteristics of HA with those of collagen, a material widely applied in bone tissue engineering with MSCs and a useful material for scaling up artificial grafts for endochondral ossification17. In an immunocompromised mouse model, HA and collagen constructs seeded with human MSCs were evaluated for in vivo ECO potential by subcutaneous implantation. The results show that HA hydrogels are excellent as a scaffold for MSCs to create artificial cartilage grafts that allow bone formation through ECO.
The protocol is divided into two steps. First, constructs of human MSCs seeded on hyaluronan hydrogel are prepared and differentiated into hypertrophic cartilage in vitro. Next, the differentiated constructs are implanted subcutaneously in a nude model to induce endochondral ossification in vivo (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25w/v% Trypsin-1mmol/L EDTA.4Na Solution</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>209-16941</td>
			<td></td>
		</tr>
		<tr>
			<td>Antisedan</td>
			<td>Nippon Zenyaku Kogyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbate-2-phosphate</td>
			<td>Nacalai Tesque</td>
			<td>13571-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Bambanker</td>
			<td>GC Lymphotec</td>
			<td>CS-02-001</td>
			<td></td>
		</tr>
		<tr>
			<td>basic fibroblastic growth factor</td>
			<td>Reprocell</td>
			<td>RCHEOT002&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>012-23881</td>
			<td>7.5 w/v%</td>
		</tr>
		<tr>
			<td>Countess Automated Cell Counter with cell counting chamber slides and Trypan Blue stain 0.4%</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>dexamethasone</td>
			<td>Merck</td>
			<td>D8893</td>
			<td></td>
		</tr>
		<tr>
			<td>Domitor</td>
			<td>Nippon Zenyaku Kogyo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dormicum</td>
			<td>Astellas Pharma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Merck</td>
			<td>D6429</td>
			<td>high glucose</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium/Nutrient Mixture F-12 Ham</td>
			<td>Merck</td>
			<td>D6421</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Hyclone</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin sulfate</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>1676045</td>
			<td>&#160;10 mg/mL</td>
		</tr>
		<tr>
			<td>Haccpper Generator</td>
			<td>TechnoMax</td>
			<td>CH-400-5QB</td>
			<td>50 ppm hypochlorous acid water</td>
		</tr>
		<tr>
			<td>Human Mesenchymal Stem Cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>HyStem Cell Culture Scaffold Kit</td>
			<td>Merck</td>
			<td>HYS020</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#223;</td>
			<td>PeproTech</td>
			<td>AF-200-01B</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS-G supplement</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>090-06741</td>
			<td>&#215;100</td>
		</tr>
		<tr>
			<td>L-Alanyl-L-Glutamine</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>016-21841</td>
			<td>200mmol/L (&#215;100)</td>
		</tr>
		<tr>
			<td>L-proline</td>
			<td>Nacalai Tesque</td>
			<td>29001-42</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Thyroxine</td>
			<td>Merck</td>
			<td>T1775</td>
			<td></td>
		</tr>
		<tr>
			<td>MSCGM Mesenchymal Stem Cell Growth Medium 
			BulletKit</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>paraffin</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>165-13375</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS / pH7.4 100ml</td>
			<td>Medicago</td>
			<td>09-2051-100</td>
			<td></td>
		</tr>
		<tr>
			<td>TGF-&#946;3&#160;</td>
			<td>Proteintech</td>
			<td>HZ-1090</td>
			<td></td>
		</tr>
		<tr>
			<td>Vetorphale</td>
			<td>Meiji Seika Kaisha</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visiocare Ointment</td>
			<td>SAVAVET/SAVA Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>FUJIFILM Wako Pure Chemical&#160;</td>
			<td>048-34332</td>
			<td></td>
		</tr>
	</tbody>
</table>

integrated bone formation through in vivo endochondral ossification using mesenchymal stem cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it does not have a mechanism to induce angiogenesis. Implanting artificial cartilage tissue fabricated from MSCs induces angiogenesis and bone formation in vivo via endochondral ossification (ECO). Therefore, this ECO-mediated approach may be a promising bone regeneration therapy in the future. An important aspect of the clinical application of this ECO-mediated approach is establishing a protocol for preparing enough cartilage to be implanted to repair the bone defect. It is especially not practical to design a single mass of grafted cartilage of a size that conforms to the shape of the actual bone defect. Therefore, the cartilage to be transplanted must have the property of forming bone integrally when multiple pieces are implanted. Hydrogels may be an attractive tool for scaling up tissue-engineered grafts for endochondral ossification to meet clinical requirements. Although many naturally derived hydrogels support MSC cartilage formation in vitro and ECO in vivo, the optimal scaffold material to meet the needs of clinical applications has yet to be determined. Hyaluronic acid (HA) is a crucial component of the cartilage extracellular matrix and is a biodegradable and biocompatible polysaccharide. Here, we show that HA hydrogels have excellent properties to support in vitro differentiation of MSC-based cartilage tissue and promote endochondral bone formation in vivo.

MSC-encapsulated HA hydrogels were cultured in chondrogenic medium supplemented with TGF&#946;3, an inducer of chondrogenesis41 (step 4.1). We compared the properties of HA with those of collagen, which has been shown to be effective in creating MSC-based artificial cartilage grafts for endochondral ossification, as described previously38. Undifferentiated MSCs were not included as negative controls in this study because it has been demonstrated that undifferentiated MSCs require mineralized surfaces as a priming substrate to generate bone by osteogenic differentiation (i.e., intramembrane ossification)7.
The size of the hyaluronan hydrogels did not change throughout the in vitro culture period, as judged based on diameters measured under an inverted microscope, whereas the collagen hydrogels used for comparison began to shrink soon after the culture started. To reduce the size difference between the HA and collagen constructs after in vitro culture, the number of MSCs and the volume of the gel used to create the HA hydrogels were halved compared to those used for the collagen hydrogels. However, the wet weight of the HA constructs after 3 weeks of cartilage culture was 4x heavier than that of the collagen constructs.
Chondrogenic and hypertrophic differentiation in vitro. 
After chondrogenic differentiation (at week 3 of in vitro culture), sulfated glycosaminoglycans (sGAG)-positive (Safranin O/fast green staining; Figure 3A) and type II collagen-positive extracellular matrix was observed in both constructs, indicating that both HA and collagen hydrogels supported chondrogenesis. However, compared to the collagen construct, the distribution of sGAG, type II collagen (Col II), and type X collagen (Col X) were more homogeneous throughout the tissue in the HA constructs. Differences between the two constructs were also apparent in cell morphology: cells with a chondrocyte-like rounded morphology were distributed throughout the tissue in the HA constructs. In the collagen constructs, however, cells in the periphery showed a heterogeneous morphology, ranging from irregular/stretched to rounded morphology. Consistent with this, the average roundness of cells in the HA constructs was higher than in the collagen constructs, both in the periphery and central regions (34% versus. 77.6% (p&#60;0.01) and 78.3% vs. 100% (p&#60;0.05), respectively; Figure 3B). At 5 weeks of culture, calcium deposition was detected at the outer edges in both constructs (alizarin red; Figure 3C). Furthermore, expression of chondrogenic (Sox-9, aggrecan (ACAN), Col II), hypertrophic (Col X, matrix metallopeptidase 13 (MMP-13)), and osteogenic (type I collagen (Col I), bone sialoprotein (BSP), osteocalcin (OCN)) markers14 was detected by quantitative real-time RT-PCR, confirming the progression of chondrogenic and hypertrophic differentiation during in vitro culture (Figure 4).
Endochondral ossification of subcutaneously implanted constructs in vivo. 
Subcutaneous pockets were created on the back of nude mice, and two to three constructs were implanted in each pocket after hypertrophic differentiation (at week 5 of in vitro culture). At 4 or 8 weeks after implantation, all HA constructs were attached to each other in the implanted pockets (Table 1). In the collagen constructs, adhesion was observed in 60% of the pockets, while in the remaining pockets, the grafts were independent of each other. Hematoxylin and eosin (H&#38;E) staining revealed that in both constructs at 8 weeks post-implantation, osteoid tissue with lamellar morphology was formed in the outer regions of the constructs (Figure 5Aa,f). sGAG staining disappeared in the HA constructs, indicating the loss of their cartilage phenotype (Figure 5Ah). In both constructs, a bone marrow component containing hematopoietic cells and adipose tissue was formed between the inner cartilage and the outer osteoid tissues. In all HA constructs, the bone tissues of the fused constructs were connected and surrounded by joint fibrous tissue and bone marrow developed along the space between the two adherent constructs, indicating that multiple HA constructs tend to form integrated bone tissue (Figure 5Ag). The adhered collagen constructs were similarly integrated in two of the three fused cases, but in the third case, the bone tissue of the two constructs was not connected (Table 1 and&#160;Figure 5Ab). In both constructs, vessels identified by CD31-positive endothelial cells42 were observed in the bone marrow (Figures 5Ad,i), and the inner cartilage regions were surrounded by TRAP-positive multinucleated cells of the osteoclast lineage, indicating that cartilage tissue was subject to remodeling (Figures 5Ae,j).
Mineral was deposited in the outer osteoid region in both HA and collagen constructs (Figure 5B). While the total mineral density of the new bone was similar between HA and collagen constructs, the mineral volume was significantly higher in the HA constructs than that in the collagen constructs, which may reflect the fact that HA hydrogels did not shrink during in vitro culture, resulting in larger constructs than collagen hydrogels (Figure 5C,D).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65573/65573fig01.jpg" /> 
Figure 1: Experimental design. The first step of this protocol is to encapsulate expanded MSCs in HA hydrogels to promote chondrogenic and hypertrophic differentiation in vitro. Constructs are cultured in chondrogenic differentiation conditions for 3 weeks, followed by an additional 2 weeks in hypertrophic differentiation conditions. The second step of this protocol is to subcutaneously implant the constructs into nude mice at week 5 of in vitro culture to undergo endochondral ossification in vivo for 8 weeks. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/65573/65573fig02.jpg" /> 
Figure 2: Preparation of MSCs-seeded hydrogels. Drop modified HA/crosslinker solution containing MSCs onto a paraffin-coated 24-well plate and allow to solidify at 37 &#176;C for 30 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/65573/65573fig03.jpg" /> 
Figure 3: Histological and immunohistochemical analysis of HA and collagen constructs after in vitro culture22. (A) Constructs at 3 weeks (3W) of in vitro culture were stained for sGAG (safranin O/fast green), type II collagen (Col II), and type X collagen (Col X). (B) The average circularity values of cells at 3 weeks of in vitro culture (n = 5). Open and grey bars indicate collagen and HA constructs, respectively. Groups without a common letter are statistically different (a versus b, p&#60;0.01; b versus c, p&#60;0.05). (C) Constructs at 5 weeks (5W) of in vitro culture were stained for calcium (alizarin red). All pictures were captured with the same magnification (Scale bar: 200 &#181;m). A low-magnification overview of the entire tissues is shown in the insets (Scale bar: 1 mm). Error bars represent mean &#177; standard deviation (SD). The one-way ANOVA followed by Tukey&#39;s multiple comparisons test was used to determine significant differences between groups. This figure has been modified from38. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/65573/65573fig04.jpg" /> 
Figure 4: Gene expression analysis of HA and collagen constructs at 3 and 5 weeks of in vitro culture. (A) Chondrogenic and hypertrophic markers. (B) Osteogenic markers. Values are presented as mean &#177; standard deviation (SD) (n = 3). Undifferentiated MSCs expressed high levels of Col I and MMP-13 and low levels of Sox-9 and Col X; they did not express (#) ACAN, Col II, BSP, and OCN. Error bars represent mean &#177; standard deviation (SD). This figure has been modified from38. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/65573/65573fig05.jpg" /> 
Figure 5: Immunohistochemical analysis and calcification of HA and collagen constructs post-implantation at 8 weeks. (A) Constructs were stained for Hematoxylin and Eosin (H&#38;E, a, b, f, and g), safranin O/Fast Green (c and h), endothelial cells (Cluster of Differentiation 31, CD31) (d and i), and Tartrate-resistant acid phosphatase (TRAP, e and j). (a and f) Overview of the entire tissues (scale bar = 500 &#181;m). H&#38;E, safranin O, CD31: scale bar = 200 &#181;m; TRAP: scale bar= 50 &#181;m. Arrows indicate CD31-positive endothelial cells. Abbreviations: c = inner cartilage tissue; o = outer osteoid tissue. (B) MicroCT (&#181;CT) imaging of collagen and HA constructs (main and inset image scale bars = 2 mm). (C,D) The total mineral density (C) and volume (D) of HA and collagen constructs (n = 4). ** indicates significant differences; p &#60;0.01. Four constructs were analyzed per group using &#181;CT. Error bars represent mean &#177; standard deviation (SD). Student&#39;s t-test was used to determine significant differences between groups. This figure has been modified from38. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/65573/65573fig06.jpg" /> 
Figure 6: Strategy for facilitating ECOs using HA-based artificial cartilage. Schematic diagram of ossification and bone marrow development without and with fragmentation (&#181;-pellets) of implanted HA constructs. (A) HA hydrogels have an excellent fusion tendency to form an integrated bone with vascularization and marrow development between fused grafts in vivo. The resulting implanted tissue, however, remained predominantly in a cartilage state even 8 weeks post-implantation. (B) Given the tendency of HA constructs to fuse to form integrated bone, processing HA constructs into &#181;-pellets may accelerate the remodeling rate of transplanted tissue and promote bone formation. Micronizing the constructs increases surface area, which may promote the resorption of cartilage tissue and the formation of bone tissue and may also promote angiogenesis and bone marrow development because of the increased space for host cell infiltration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/65573/65573fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hydrogel</td>
			<td>Experiments</td>
			<td>Adhered</td>
			<td>United</td>
			<td>% United</td>
		</tr>
		<tr>
			<td>Collagen</td>
			<td>5</td>
			<td>3</td>
			<td>2</td>
			<td>40</td>
		</tr>
		<tr>
			<td>HA</td>
			<td>5</td>
			<td>5</td>
			<td>5</td>
			<td>100</td>
		</tr>
	</tbody>
</table>
Table 1: Unification rate when multiple constructs were implanted in a single pocket. Two or three constructs were subcutaneously implanted in a single pocket. Constructs were harvested 4- or 8-weeks post-implantation and histologically examined.

Watch this Scientific Journal Video about Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration at JoVE.com

Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Conventional bone regeneration therapy using mesenchymal stem cells (MSCs) is difficult to apply to bone defects larger than the critical size because it ...

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908 Views.  Tokyo Medical and Dental University. Bone therapy via endochondral ossification by implanting artificial cartilage tissue produced from mesenchymal stem cells has the potential to circumvent the drawbacks of conventional therapies. Hyaluronic acid hydrogels are effective in scaling up uniformly differentiated cartilage grafts as well as creating integrated bone with vascularization between fused grafts in vivo.

Video: Author Spotlight: Enhancing Bone Regeneration with Vascularized Artificial Cartilage Integration 

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal stem cells, MSCs) and endothelial colony forming progenitor cells (ECFCs). Both cell types are key players in maintaining the integrity of tissue and are probably also involved in regenerative processes and tumor formation.
To study their biology and function in a comparative manner it is important to have both cells types available from the same donor. It may also be beneficial for regenerative purposes to derive MSCs and ECFCs from the same tissue.
Because cellular therapeutics should eventually find their way from bench to bedside we established a new method to isolate and further expand progenitor cells without the use of animal protein. Pooled human platelet lysate (pHPL) replaced fetal bovine serum in all steps of our protocol to completely avoid contact of the cells to xenogeneic proteins.
This video demonstrates a methodology for the isolation and expansion of progenitor cells from one umbilical cord.
All materials and procedures will be described.

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells MSCs and Endothelial Colony Forming Progenitor Cells ECFCs

This protocol describes the isolation and subsequent expansion of mesenchymal stromal cells and endothelial colony forming cells without the use of animal serum to generate autologous pairs for experimental transplantation purposes.

The umbilical cord is a rich source for progenitor cells with high proliferative potential including mesenchymal stromal cells (also termed mesenchymal ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

Isolation &#38; Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor formation. Taken together these studies suggest that resident lung MSC play a role during pulmonary tissue homeostasis, injury and repair during diseases such as pulmonary fibrosis (PF) and arterial hypertension (PAH). Here we describe a technology to define a population of resident lung MSC. The definition of this population in vivo pulmonary tissue using a define set of markers facilitates the repeated isolation of a well-characterized stem cell population by flow cytometry and the study of a specific cell type and function.

Isolation & Characterization of Hoechstlow CD45negative Mouse Lung Mesenchymal Stem Cells

In this article we demonstrate the isolation of murine resident lung mesenchymal stem cells (lung MSC), their expansion, characterization and analysis of immunomodulatory properties.

Tissue resident mesenchymal stem cells (MSC) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis and tumor ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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) ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

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Chapters in this video

Isolation and Animal Serum Free Expansion of Human Umbilical Cord Derived Mesenchymal Stromal Cells (MSCs) and Endothelial Colony Forming Progenitor Cells (ECFCs)

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Isolation & Characterization of Hoechst^low CD45^negative Mouse Lung Mesenchymal Stem Cells

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Induction and Analysis of Epithelial to Mesenchymal Transition

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

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

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury