This work was supported by the CIRM Early Translational II Research Award TR2-01821, NIH/NIDCR (grants R21 DE0177711 and RO1 DE01607), UC Discovery Grant 07-10677, AWJ and RKS have T32 training fellowships awards (5T32DE007296-14), JNZ has a CIRM training fellowship (TG2-01169).

1. Perivascular Stem Cell Isolation
This is described in details in the adjacent article "Purification of Perivascular Stem Cells from Human White Adipose tissue", by M. Corselli et al.
2. Scaffold Creation
<ol>
	<li>Scaffolds are custom-made per previously published protocol from poly(lactic-co-glycolic acid) (PLGA, Burmingham Polymer) with hydroxyapatite coating4-6. Apatite-coated PLGA scaffolds are fabricated from 85/15 PLGA by solvent casting and a particulate leaching process. Scaffolds are created in a spherical shape (2-mm diameter) for muscle pouch implantation, a discoid shape (4 mm in diameter) for calvarial implantation, or cylindrical (4 mm in diameter, 6 mm in length) for femoral segmental defects.</li>
	<li>Briefly, PLGA/chloroform solutions mixed with sucrose (polymer/sucrose ratio 5/95, w/w) are cast into a 200-300-&mu;m diameter Teflon mold to create the custom-made construct. After freeze-drying overnight, scaffolds are removed from the Teflon mold and immersed in ddH2O to dissolve the sucrose. Scaffolds are disinfected by immersion in 70% ethanol for 30 min, followed by three rinses of ddH2O.</li>
	<li>For apatite coating, a simulated body fluid (SBF) solution is prepared by sequentially dissolving CaCl2, MgCl2&bull;6H2O, NaHCO3, and K2HPO4&bull;3H2O in ddH2O. Solution pH is lowered to 6 by adding 1M hydrochloric acid to increase the solubility. Na2SO4, KCl, and NaCl are added and the final pH is adjusted to 6.5 (SBF 1).</li>
	<li>Mg2+ and HCO3- free SBF (SBF 2) is prepared by adding CaCl2 and K2HPO4&bull;3H2O in ddH2O and pH is lowered to 6. KCl and NaCl are added and the final pH is adjusted to 6.8. All solutions are sterile filtered through a 0.22 &mu;m PES membrane (Nalgene). Immediately prior to the coating process, the dried PLGA scaffolds are subjected to glow discharge argon plasma etching (Harrick Scientific) to improve wetting and coating uniformity.</li>
	<li>Etched scaffolds are then incubated in SBF 1 for 12 h and changed to Mg2+ and HCO3- free SBF 2 for another 12 h at 37 &deg;C under gentle stirring. Coated scaffolds are washed with ddH2O to remove excess ions and lyophilized prior to further studies.</li>
</ol>
3. The Muscle Pouch Model Implantation
<ol>
	<li>100 &mu;l of a PSC suspension in PBS (phosphate buffered saline) are gently dropped onto the spherical PLGA-based implant immediately prior to implantation. Cells have been pre-labeled by lentiviral insertion of Firefly luciferase, so as to permit in vivo tracking post implantation. Cell density is 2.5 x 105 per implant.</li>
	<li>SCID (severe combined immunodeficient) mice are used at 6 weeks of age. Animals are anesthetized by isoflurane inhalation and premedicated with buprenorphine (Bedford Labs). After standard Betadine preparation, bilateral incisions in the hindlimbs are made (longitudinal and 2 mm in length).</li>
	<li>Pockets are cut in the biceps femoris muscles by blunt dissection parallel to the muscle fiber long axis. For each mouse, the PLGA-based implant with PSCs is inserted, and the fascia overlying the muscle is sutured with 5-0 Vicryl (Ethicon).</li>
	<li>The skin is next closed with 5-0 Vicryl in a subcuticular pattern. Animals are treated postoperatively with buprenorphine for 48 hours and TMP/SMX (Trimethoprim/ Sulfamethoxazole; Qualitest) for 10 days.</li>
</ol>
4. The Calvarial Defect Model Implantation
<ol>
	<li>After isoflurane anesthesia of SCID mice (12-14 weeks old), the hair is clipped and the skin disinfected with betadine per protocol.</li>
	<li>A 8-mm skin incision is made along the mid-sagittal suture of the mouse calvarium. Next, the calvarial periosteum is gently removed by Q-tip application.</li>
	<li>Next, using diamond-coated trephine bits in a high-speed dental drill, a 4-mm parietal bone defect is created. The defect is full thickness - however care is taken not to injure the underlying dura mater.</li>
	<li>The custom-made PLGA-based implant with engrafted PSCs is then placed gently in the defect site. Cell density is 2.5 x 105 per implant. Finally, the skin is sutured with 6-0 Vicryl. Animals are treated postoperatively with buprenorphine for 48 hours and TMP/SMX for 10 days.</li>
</ol>
5. The Femoral Segmental Defect Model Implantation
<ol>
	<li>Athymic rats (12-14 weeks old) are anesthetized under isoflurane inhalation. The femur is scrubbed and prepared per standard protocol with betadine (Figure 1).</li>
	<li>A 27-30-mm longitudinal incision is made over the anterolateral aspect of the femur. The femoral shaft is then exposed by separating the vastus lateralis and biceps femoris muscles (Figure 2).</li>
	<li>To maximize consistency of bone regeneration, the periosteum overlying the femoral defect is completely removed with the resected femoral segment.</li>
	<li>A polyethylene plate (length, 23 mm; width, 4 mm; height, 4 mm) is placed on the anterolateral surface of the femur. The plate contains six pre-drilled holes to accommodate 0.9 mm diameter threaded Kirschner wires (Zimmer). Taking the plate as a template, six threaded Kirschner wires are drilled through the plate and both cortices (Figure 3).</li>
	<li>Next, with a small oscillating saw blade (Stryker, MI), a 6-mm mid-diaphyseal defect is created. Segmental defects are then treated by the insertion of PLGA based implants which have been laden with cells as per protocol (Figure 4).</li>
	<li>The overlying muscle and fascia are closed with 4-0 Vicryl absorbable sutures to secure the implant in place, and the skin is sutured.</li>
</ol>
6. In Vivo Assessments
<ol>
	<li>Radiographic assessments are performed in a longitudinal manner by both high resolution XR and high resolution &mu;CT (micro computed tomography) analysis. For &mu;CT analysis (Skyscan 1172F), images are scanned at a resolution of 19.73 &mu;m (100 kV and 100 mA radiation source, using a 0.5 mm aluminum filter). Images are analyzed using DataViewer, Recon, CTAn, and CTVol softwares.</li>
	<li>Bioluminescence imaging is also done in a serial manner to assess cell engraftment, viability, proliferation and exclude migration out of the implant site. Bioluminescence imaging is performed using an IVIS Lumina II device (Caliper Life Sciences). Light outputs are quantified using Living Image software (Xenogen). Total light output is recorded in photons/second/sq cm/steradian.</li>
	<li>Histological and histomorphometric analysis is performed postmortem. Routine stains employed include Masson's trichrome, aniline blue, pentachrome, and Picrosirius red. Histomorphometric analysis is performed easily with either aniline blue or pentachrome stains, in which osteoid appears dark blue and yellow, respectively. Pixels per high powered field are calculated using the magic wand tool in Adobe Photoshop.</li>
</ol>
7. Representative Results
As both the calvarial and femoral defects are critical-sized, no significant healing should be expected without treatment with growth factors or exogenous stem cells.
In terms of surgical maneuvers, the muscle pouch dissection should be along fascial planes and thus minimal bleeding should be encountered. Even though the muscle pouch model is performed bilaterally, the mouse should be walking with ease on postoperative day 1. For the calvarial defect, bleeding is encountered but can be soaked with a Q-tip. Extreme care should be taken not to injure the underlying dura mater - as this will interfere with normal healing. For the FSD model, care is taken not to injure the major blood vessels so as not to cause excessive bleeding or the femoral nerve to prevent neurologic damage. Kirschner wires are drilled with gentle pressure so as not to damage the cortical bone in the process.
<img alt="Figure 1." src="/files/ftp_upload/2952/2952fig1.jpg" /> 
Figure 1. Preoperative preparation for Femoral Segmental Defect (FSD) in Athymic Rats. Male rats (12-14 weeks old) are anesthetized under isoflurane inhalation. The femur is scrubbed and prepped per standard protocol with betadine.
<img alt="Figure 2." src="/files/ftp_upload/2952/2952fig2.jpg" /> 
Figure 2. Surgical exposure for Femoral Segmental Defect (FSD) creation. A 27-30 mm longitudinal incision is made over the anterolateral aspect of the femur. The lateral aspect of the femoral shaft is then exposed by separating the vastus lateralis and biceps femoris muscles.
<img alt="Figure 3." src="/files/ftp_upload/2952/2952fig3.jpg" /> 
Figure 3. Fixation for Femoral Segmental Defect (FSD) creation. A polyethylene plate (length, 23 mm; width, 4 mm; height, 4 mm) is placed on the anterolateral surface of the femur. The plate contains six pre-drilled holes to accommodate 0.9 mm diameter threaded Kirschner wires. Taking the plate as a template, six threaded Kirschner wires are drilled through the plate and both cortices. Next, a 6 mm mid-diaphyseal defect is created. Once this is performed, a custom-made scaffold is directly inserted which exactly fits the defect site (not shown).
<img alt="Figure 4." src="/files/ftp_upload/2952/2952fig4.jpg" /> 
Figure 4. Example of Calvarial Defect Postoperative. A 4 mm, circular calvarial defect is created in the right parietal bone in athymic mice. Imaged here is a defect site implanted with PSCs at 8 weeks postoperative. Note the presence of new bone within the defect site.

<ol>
	<li>Crisan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perivascular+origin+for+mesenchymal+stem+cells+in+multiple+human+organs.">A perivascular origin for mesenchymal stem cells in multiple human organs.</a> Cell Stem Cell. 3, 01-13 (2008).</li><li>Chen, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+multi-lineage+progenitor+cells+in+human+organs:+regenerative+units,+cytokine+sources+or+both.">Perivascular multi-lineage progenitor cells in human organs: regenerative units, cytokine sources or both.</a> Cytokine Growth Factor Rev. 20, 429-434 (2009).</li><li>Corselli, M., Chen, C. W., Crisan, M., Lazzari, L., Peault, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perivascular+ancestors+of+adult+multipotent+stem+cells.">Perivascular ancestors of adult multipotent stem cells.</a> Arterioscler. Thromb Vasc. Biol. 30, 1104-1104 (2010).</li><li>Zara, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nell-1+enhances+bone+regeneration+in+a+rat+critical+sized+femoral+segmental+defect+model.">Nell-1 enhances bone regeneration in a rat critical sized femoral segmental defect model.</a> Plast. Reconstr. Surg. , (2010).</li><li>James, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deleterious+Effects+of+Freezing+on+Osteogenic+Differentiation+of+Human+Adipose-Derived+Stromal+Cells+In+Vitro+and+In+Vivo.">Deleterious Effects of Freezing on Osteogenic Differentiation of Human Adipose-Derived Stromal Cells In Vitro and In Vivo.</a> Stem Cells Dev. 20, 427-439 (2011).</li><li>Lee, M., Chen, T. T., Iruela-Arispeb, M. L., Wu, B. M., Dunn, J. C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+protein+delivery+from+modular+polymer+scaffolds.">Modulation of protein delivery from modular polymer scaffolds.</a> Biomaterials. 28, 1862-1870 (2007).</li><li>Aghaloo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+the+role+of+nell-1+gene+modified+goat+bone+marrow+stromal+cells+in+promoting+new+bone+formation.">A study of the role of nell-1 gene modified goat bone marrow stromal cells in promoting new bone formation.</a> Mol. Ther. 15, 1872-1880 (2007).</li><li>Levi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Adipose-Derived+Stromal+Cells+Stimulate+Autogenous+Skeletal+Repair+via+Paracrine+Hedgehog+Signaling+with+Calvarial+Osteoblasts.">Human Adipose-Derived Stromal Cells Stimulate Autogenous Skeletal Repair via Paracrine Hedgehog Signaling with Calvarial Osteoblasts.</a> Stem Cells Dev. 20, 243-257 (2011).</li><li>Leucht, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+origin+and+Hox+status+determine+progenitor+cell+fate+during+adult+bone+regeneration.">Embryonic origin and Hox status determine progenitor cell fate during adult bone regeneration.</a> Development. 135, 2845-2854 (2008).</li><li>Rajashekhar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFATS+collection:+Adipose+stromal+cell+differentiation+is+reduced+by+endothelial+cell+contact+and+paracrine+communication:+role+of+canonical+Wnt+signaling.">IFATS collection: Adipose stromal cell differentiation is reduced by endothelial cell contact and paracrine communication: role of canonical Wnt signaling.</a> Stem Cells. 26, 2674-2681 (2008).</li><li>Zuk, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+adipose+tissue+is+a+source+of+multipotent+stem+cells.">Human adipose tissue is a source of multipotent stem cells.</a> Mol Biol Cell. 13, 4279-4295 (2002).</li></ol>

K.T, B.P., and C.S. are inventors of perivascular stem cell-related patents filed from UCLA. Drs. K.T, and C.S. are founders of Scarless Laboratories Inc. which sublicenses perivascular stem cell-related patents from the UC Regents. Dr. Chia Soo is also an officer of Scarless Laboratories, Inc.

The isolation of PSCs is well described elsewhere1-3, including a separately submitted JoVE publication specifically addressing PSC isolation protocols and methods. The specific purpose of this article is to describe and demonstrate 3 protocols for PSC in vivo application for bone formation/regeneration. The SCID mouse muscle pouch is a commonly described model for ectopic human bone formation7. Important differences exist between ectopic and orthotopic (defect) models for bone, including paracrine interaction with host bone-forming cells8 as well as an abundance of osteogenic signaling factors present in the skeletal defect microenvironment. Two defects are presented here, a calvarial defect8 and femoral segmental defect4. Both are well-documented to be critical sized (i.e. will not heal on their own). 
Interesting differences exist between calvarial and femoral defects. First, the cell:cell interaction between xenografted PSCs and endogenous cells is very different. In terms of a calvarial defect, PSCs interact with the underlying dura mater (the outermost layer of the meninges), as well as those osteoblasts and periosteal cells circumscribing the defect site. Importantly, the interaction between implanted cells and surrounding osteoblasts8, or implanted cells and underlying dura (Levi et al., in press) are critical for normal stem cell mediated osteogenesis to proceed. In terms of the femoral segmental defect (FSD), xenografted PSCs are exposed to a very different cell and cytokine environment. For example, the FSD site is composed of the marrow and accompanying mesenchymal stem cells, as well as the endosteum, periosteum and long-bone osteoblasts. Theoretically, each cell has its own reaction to injury, and each may have cell:cell interactions with PSC xenografts.
Other clear differences exist between calvarial and femoral defects. The calvarial bones initially form through intramembranous ossification, while the long bones form through a cartilage intermediate (endochondral ossification). Moreover, the reparative process post-injury also mimics these developmental origins. Post-FSD, cartilage callus formation is observed, whereas no cartilage intermediate is formed within a parietal bone defect. Finally, the embryonic origin of the skull may differ from that of the long bones. The majority of the skull (including perivascular cells &#x2013; pericytes &#x2013; in the whole head region) is derived from the neural crest (mesectoderm), while the appendicular skeleton is of paraxial mesoderm derivation9. All these differences may result in significant differences in terms of PSC-mediated bone repair.
 The use of PSCs has several benefits over traditional adipose-derived stromal cells (ASCs). PSCs do not require culture and are a purified cell population which does not include other stromal cells that do not participate in &#x2013; and can even negatively regulate - osteogenic differentiation, such as endothelial cells10. In contrast, for example, clonal analyses of ASCs show that only a subpopulation are capable of undergoing osteogenic differentiation in vitro11. Ultimately, skeletal tissue engineering efforts will likely incorporate an osteocompetent stem cell (such as PSCs) with exogenous growth factors and an osteoconductive scaffold (such as HA-PLGA used in the present methods) so as to best heal skeletal defects.

use of human perivascular stem cells for bone regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Watch this Scientific Journal Video about Use of Human Perivascular Stem Cells for Bone Regeneration at JoVE.com

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

use-of-human-perivascular-stem-cells-for-bone-regeneration

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Bioengineering

Unit 1

Regeneration and Repair

SCIENTISTS IN ACTION

20.5K Views.  School of Dentistry, UCLA. Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Video: Use of Human Perivascular Stem Cells for Bone Regeneration

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

Transplantation of Induced Pluripotent Stem Cell-derived Mesoangioblast-like Myogenic Progenitors in Mouse Models of Muscle Regeneration

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells similar to pericyte-derived mesoangioblasts (iPSC-derived mesoangioblast-like stem/progenitor cells: IDEMs) can be established from iPSCs generated from patients affected by different forms of muscular dystrophy. Patient-specific IDEMs can be genetically corrected with different strategies (e.g. lentiviral vectors, human artificial chromosomes) and enhanced in their myogenic differentiation potential upon overexpression of the myogenesis regulator MyoD. This myogenic potential is then assessed in vitro with specific differentiation assays and analyzed by immunofluorescence. The regenerative potential of IDEMs is further evaluated in vivo, upon intramuscular and intra-arterial transplantation in two representative mouse models displaying acute and chronic muscle regeneration. The contribution of IDEMs to the host skeletal muscle is then confirmed by different functional tests in transplanted mice. In particular, the amelioration of the motor capacity of the animals is studied with treadmill tests. Cell engraftment and differentiation are then assessed by a number of histological and immunofluorescence assays on transplanted muscles. Overall, this paper describes the assays and tools currently utilized to evaluate the differentiation capacity of IDEMs, focusing on the transplantation methods and subsequent outcome measures to analyze the efficacy of cell transplantation.

Induced pluripotent stem cell (iPSC)-derived myogenic progenitors are promising candidates for cell therapy strategies to treat muscular dystrophies. This protocol describes transplantation and functional measurements required to evaluate the engraftment and differentiation of iPSC-derived mesoangioblasts (a type of muscle progenitors) in mouse models of acute and chronic muscle regeneration.

Patient-derived iPSCs could be an invaluable source of cells for future autologous cell therapy protocols. iPSC-derived myogenic stem/progenitor cells ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Biological Compatibility Profile on Biomaterials for Bone Regeneration

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such lesions, there are potential complications with their use. Thus, material scientists are developing synthetic, biocompatible biomaterials to overcome these problems. In this study, we present a multidisciplinary platform for evaluating biomaterials for bone repair. We combined expertise from bone biology and immunology to develop a platform including in vitro osteoclast (OC) and osteoblast (OB) assays and in vivo mouse models of bone repair, immunogenicity, and allergenicity. We demonstrate how to perform the experiments, summarize the results, and report on biomaterial biocompatibility. In particular, we tested OB viability, differentiation, and mineralization and OC viability and differentiation in the context of &#946;-tricalcium phosphate (&#946;-TCP) disks. We also tested a &#946;-TCP/Collagen (&#946;-TCP/C) foam which is a commercially available material used clinically for bone repair in a critical-sized calvarial bone defect mouse model to determine the effects on the early phase of bone healing. In parallel experiments, we evaluated immune and allergic responses in mice. Our approach generates a biological compatibility profile of a bone biomaterial with a range of parameters necessary for predicting the biocompatibility of biomaterials used for bone healing and repair in patients.

The number of novel biomaterials engineered for repairing large bone lesions is continuously expanding with the aim to enhance bone healing and overcome the complications associated with bone transplantation. Here, we present a multidisciplinary strategy for pre-clinical biocompatibility testing of biomaterials for bone repair.

Large non-union bone fractures are a significant challenge in orthopedic surgery. Although auto- and allogeneic bone grafts are excellent for healing such ...

Uptake of New Lipid-coated Nanoparticles Containing Falcarindiol by Human Mesenchymal Stem Cells

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and size by low-density lipoproteins (LDLs) because cancer cells have an increased need for cholesterol to proliferate, and this has been exploited as a mechanism for delivering anticancer drugs to cancer cells. Moreover, depending on drug chemistry, encapsulating the drug can be advantageous to avoid degradation of the drug during circulation in vivo. Therefore, in this&#160;study, this design is used to fabricate lipid-coated nanoparticles of the anticancer drug falcarindiol, providing a potential new delivery system of falcarindiol in order to stabilize its chemical structure against degradation and improve its uptake by tumors. Falcarindiol nanoparticles, with a phospholipid and cholesterol monolayer encapsulating the purified drug core of the particle, were designed. The lipid monolayer coating consists of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol (Chol), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE PEG 2000) along with the fluorescent label DiI (molar ratios of 43:50:5:2). The nanoparticles are fabricated using the rapid injection method, which is a fast and simple technique to precipitate nanoparticles by good-solvent for anti-solvent exchange. It consists of a rapid injection of an ethanol solution containing the nanoparticle components into an aqueous phase. The size of the fluorescent nanoparticles is measured using dynamic light scattering (DLS) at 74.1 &#177; 6.7 nm. The uptake of the nanoparticles is tested in human mesenchymal stem cells (hMSCs) and imaged using fluorescence and confocal microscopy. The uptake of the nanoparticles is observed in hMSCs, suggesting the potential for such a stable drug delivery system for falcarindiol.

This article describes the encapsulation of falcarindiol in lipid-coated 74 nm nanoparticles. The cellular uptake of the nanoparticles&#160;by human stem cells into lipid droplets is monitored by fluorescent and confocal imaging. Nanoparticles are fabricated by the rapid injection method of solvent shifting, and their size is measured with the dynamic light scattering technique.

Nanoparticles are the focus of an increased interest in drug delivery systems for cancer therapy. Lipid-coated nanoparticles are inspired in structure and ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Single-Cell Optical Action Potential Measurement in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically carried out in low throughput. Rapid and ongoing expansion of induced pluripotent stem cell (iPSC) technology presents a new standard in cardiovascular research and alternate methods are now necessary to increase throughput of electrophysiological data at a single cell level. VF2.1Cl is a recently derived voltage sensitive dye which provides a rapid single channel, high magnitude response to fluctuations in membrane potential. It possesses kinetics superior to those of other existing voltage indicators and makes available functional data equivalent to that of traditional microelectrode techniques. Here, we demonstrate simplified, non-invasive action potential characterization in externally paced human iPSC derived cardiomyocytes using a modular and highly affordable photometry system.

Here we describe optical acquisition and characterization of action potentials from induced pluripotent stem cell derived cardiomyocytes using a high-speed modular photometry system.

Conventional intracellular microelectrode techniques to quantify cardiomyocyte electrophysiology are extremely complex, labor intensive, and typically ...

A Culture Method to Maintain Quiescent Human Hematopoietic Stem Cells

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, unlike more differentiated progenitors, and enter the cell cycle rapidly after chemotherapy or irradiation to treat bone marrow injury or in vitro culture. By mimicking the bone marrow microenvironment in the presence of abundant fatty acids, cholesterol, low concentration of cytokines, and hypoxia, human HSCs maintain quiescence in vitro. Here, a detailed protocol for maintaining functional HSCs in the quiescent state in vitro is described. This method will enable studies of the behavior of human HSCs under physiological conditions.

This protocol enables the maintenance of quiescent human hematopoietic stem cells in vitro by mimicking the bone marrow microenvironment using abundant fatty acids, cholesterol, lower concentrations of cytokines, and hypoxia.

Human hematopoietic stem cells (HSCs), like other mammalian HSCs, maintain lifelong hematopoiesis in the bone marrow. HSCs remain quiescent in vivo, ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...