Name: isolation of mesenchymal stem cells from human alveolar periosteum and effects of vitamin d on osteogenic activity of periosteum-derived cells
Uploaded: 2018-05-04T12:30:00
Description: Watch this Scientific Journal Video about Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells at JoVE.com

The study protocol was approved by the Institutional Review Board for Clinical Research of Chang Gung Memorial Hospital (IRB99-1828B, 100-3019C, 99-3814B, 102-1619C, 101-4728B, and 103-4223C). This study was supported by Chang Gung Memorial Hospital (CMRPG392071, CMRPG3A1141, CMRPG3A1142, and NMRPG3C0151). This manuscript was edited by Wallace Academic Editing.

The study protocol was approved by the Institutional Review Board of Chang Gung Memorial Hospital. All participants provided written informed consent.
1. Tissue Preparation
<ol>
	<li>Harvest the periosteal tissues from patients during dental surgery (Figure 1). After flap reflection under local anesthesia, take a piece of periosteum tissue from the alveolar bone using a periosteal separator31.</li>
	<li>After harvesting, store the periost...

A recently developed therapeutic modality, namely tissue engineering entailing MSCs, has numerous advantages. MSCs, which are present in several tissue types, are multipotent cells that can differentiate into a variety of functional mesodermal tissue cells37 and other cells such as osteoblasts.
The periosteum serves as a niche for progenitor cells and as a rich vasculature supply for the bone it envelops38. In our study, of the 34 investigated sa...

Although numerous relevant techniques have been developed in recent years, bone reconstruction remains limited by multiple constraints, and estimating the extent of necessary reconstruction is often impossible. Hard-tissue augmentation is required to achieve both esthetic and functional goals in addition to a favorable long-term success rate. Methods commonly used for such procedures include autogenous and allogenic bone grafting, xenografting, and alloplastic bone grafting. Among the various types of bone graft, autogenous bone grafts are considered the most effective. However, donor site morbidity, compromised vascularity, and limited tissue availability1 have been major drawbacks for autogenous bone grafting. In addition, allogenic bone grafts and xenografts have been associated with disease transmission. Currently, synthetic bone grafts are widely used to resolve these problems. However, with their lack of osteogenic potential, clinical outcomes have varied widely. Materials such as cellulose are associated with volume fluctuation, infection, and a lack of strength.
Bone augmentation using tissue engineering has generated considerable interest. In this technique, mesenchymal stem cells (MSCs) are initially used to promote osteoblast differentiation, which are then transplanted to the site of bone loss to achieve bone repair. This procedure is currently applied in cell therapy. Achieving bone reconstruction by extracting a limited amount of tissue is simpler and less invasive compared with other methods.
The potential role of MSCs as a tool for cell-based therapies aimed at dental regeneration is an emerging interest among various research groups. Studies have confirmed that MSCs can be differentiated from the following types of tissue: bone marrow, adipose, synovial membrane, pericyte, trabecular bone, human umbilical cord, and dental tissues2,3. Common sources of MSCs include bone marrow, adipose tissue, and dental tissues. Compared with MSCs derived from adipose tissue and bone marrow, the advantages of dental stem cells are easy accessibility and less morbidity after harvesting. Compared with embryonic stem cells, MSCs derived from dental tissue appear nonimmunogenic and are not associated with complex ethical concerns3.
In 2006, the International Society for Cellular Therapy recommended using the following standards to identify MSCs: First, MSCs must be capable of attaching to plastic. Second, MSCs must be positive for the surface antigens CD105, CD73, and CD90 and negative for the markers for monocytes, macrophages, and B cells in addition to the hematopoietic antigens CD45 and CD344. As a final criterion, MSCs must be able to differentiate into the following three types of cells under standard conditions of in vitro differentiation: osteoblasts, adipocytes, and chondrocytes4. To date, six types of human dental stem cell have been isolated and characterized. The first type was isolated from human pulp tissue and termed postnatal dental pulp stem cells5. Subsequently, three additional types of dental MSCs have been isolated and characterized: stem cells from exfoliated deciduous teeth6, the periodontal ligament7, and the apical papilla8. More recently, dental follicle-derived9, gingival tissue-derived10, dental bud stem cells(DBSCs)11, and periapical cyst MSCs (hPCy-MSCs)12 have also been identified.
Friedenstein was the first to define MSCs13. MSCs exhibit a high proliferation potential and can be manipulated to differentiate before being transplanted, which suggests that they are ideal candidates for regenerative procedures10.
Although most studies have used bone marrow as a source of stem cells, periosteum-derived cells (PDCs) have also been used recently14. The periosteum is more easily accessible than is the bone marrow. Therefore, in this technique, we use alveolar periosteum to eliminate the need for additional incisions during surgery and to reduce postsurgical morbidity in patients. The periosteum is the connective tissue that forms the outer lining of long bones and comprises two distinct layers: the outer fibrous layer composed of fibroblasts, collagen, and elastic fibers15, and the inner cell-rich cambium layer in direct contact with the bone surface. The cambium layer contains a mixed cell population, primarily fibroblasts16, osteoblasts17, pericytes18, and a critical subpopulation identified as MSCs19,20,21. Most studies have reported that PDCs are comparable, if not superior, to bone marrow-derived stem cells (bMSCs) in bone healing and regeneration22,23,24. The periosteum is easily accessible and exhibits excellent regenerative effectiveness. However, few studies have focused on the periosteum25,26,27.
Regarding bone repair, the current clinical practice involves the transplantation of periosteal progenitor cells amplified within supportive scaffolds. Recent studies have focused on acquiring stem cells in defective regions and employing progenitor cells for tissue regeneration20. Dentists also anticipate future application of periodontal bone regeneration in periodontal treatments and dental implants. Regarding the donor site, the periosteum can be easily harvested by general dental surgeons. This compares favorably against marrow stromal cells, as the periosteum can be accessed during routine oral surgery. Thus, the objective of this study is to establish a protocol for harvesting PDCs and to assess the morphology, attachment, viability, and proliferation of human periosteum stem cells.
Vitamin D metabolites affect the in vivo bone-mineral dynamic equilibrium. One study reported that the 24R,25-(OH)2D3 active form of Vitamin D is essential for the osteoblastic differentiation of human MSCs (hMSCs)28. Bone homeostasis and repair are regulated by a network of Vitamin D3 metabolites, of which 1,25-(OH)2D3 (calcitriol) is the most biologically active and relevant in the regulation of bone health. Vitamin D3 is essential for calcification29. In one study using 2-d-old Kunming white mice, the embryoid bodies in the mice indicated that Vitamin C and Vitamin D supplements effectively promoted the differentiation of ESC-derived osteoblasts30. Among its other biological activities, 1,25-(OH)2D3 stimulates the in vitro differentiation of hMSCs to osteoblasts, which can be monitored based on the increase in alkaline phosphatase (ALP) enzyme activity or OCN gene expression.
Few studies have detected a dose-response relationship of combined treatments with Vitamin C and 1,25-(OH)2D3 in human PDCs with a particular focus on bone tissue engineering. Therefore, in this study, we examine the optimal concentrations for single or combined treatment of 1,25-(OH)2D3 and Vitamin C for inducing osteogenic differentiation of human PDCs. The goal of this protocol is to determine whether a cell population isolated from the dental alveolar periosteum contains cells with an MSC phenotype and whether these cells can be expanded in culture (in vitro) and differentiated to form the desired tissue. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes. The second part of the study evaluates the effects of Vitamin C and 10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M 1,25-(OH)2D3 on the osteogenic activity of PDCs. The primary objective of this study is to assess the functions of Vitamin C and 1,25-(OH)2D3 during the osteoblastic differentiation of PDCs by ALP activity, and pro-osteogenic genes, such as ALP, collagen-1, OCN, BSP, and CBFA1. In addition, this study determines the optimal osteoinductive conditions for human PDCs based on these findings.

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>2-phospho-L-ascorbic acidtrisodium salt</td>
			<td>Sigma</td>
			<td>49752</td>
			<td></td>
		</tr>
		<tr>
			<td>35-mm culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>3-isobutyl-1-methylxanthine</td>
			<td>Sigma</td>
			<td>I5879</td>
			<td></td>
		</tr>
		<tr>
			<td>6&#160; well plate</td>
			<td>Corning</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>ABI</td>
			<td>Hs01029144_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase Activity Colorimetric Assay Kit</td>
			<td>BioVision</td>
			<td>K412-500</td>
			<td></td>
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		<tr>
			<td>avian myeloblastosis virus reverse transcriptase</td>
			<td>Roche</td>
			<td>10109118001</td>
			<td></td>
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		<tr>
			<td>CD146</td>
			<td>BD</td>
			<td>561013</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19</td>
			<td>BD</td>
			<td>560994</td>
			<td></td>
		</tr>
		<tr>
			<td>CD34</td>
			<td>BD</td>
			<td>560942</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44</td>
			<td>BD</td>
			<td>561858</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45</td>
			<td>BD</td>
			<td>561088</td>
			<td></td>
		</tr>
		<tr>
			<td>CD73</td>
			<td>BD</td>
			<td>561014</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90</td>
			<td>BD</td>
			<td>561974</td>
			<td></td>
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		<tr>
			<td>Cell banker1</td>
			<td>ZEAOAQ</td>
			<td>11888</td>
			<td></td>
		</tr>
		<tr>
			<td>core binding factor alpha-1</td>
			<td>ABI</td>
			<td>Hs00231692_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>26140-079</td>
			<td></td>
		</tr>
		<tr>
			<td>GAPDH</td>
			<td>ABI</td>
			<td>Hs99999905_m1</td>
			<td></td>
		</tr>
		<tr>
			<td>HLA-DR</td>
			<td>BD</td>
			<td>562008</td>
			<td></td>
		</tr>
		<tr>
			<td>indomethacin</td>
			<td>Sigma</td>
			<td>I7378</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin</td>
			<td>sigma</td>
			<td>91077C</td>
			<td></td>
		</tr>
		<tr>
			<td>insulin&#8211;transferrin&#8211;selenium-A</td>
			<td>Sigma</td>
			<td>I1884</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp Fast 96 well reaction plate(0.1ml)</td>
			<td>Life</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroAmp optical adhesive film</td>
			<td>Life</td>
			<td>4311971</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum Essential Medium 1X Alpha Modification</td>
			<td>HyClone</td>
			<td>SH30265.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Permeabilization buffer</td>
			<td>eBioscience</td>
			<td>00-8333-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate</td>
			<td>Gibco</td>
			<td>11360070</td>
			<td></td>
		</tr>
		<tr>
			<td>STRO-1</td>
			<td>BioLegend</td>
			<td>340103</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBER Green PCR Master Mix</td>
			<td>AppliedBiosystems</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Master Mix</td>
			<td>Life</td>
			<td>4304437</td>
			<td></td>
		</tr>
		<tr>
			<td>transforming growth factor-&#946;</td>
			<td>Sigma</td>
			<td>T7039&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol reagent (for RNA isolation)</td>
			<td>Life</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-glycerophosphate</td>
			<td>Sigma</td>
			<td>G9422</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">collagen-1</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39; CCTCAAGGGCTCCAACGAG-3</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-TCAATCACTGTCTTGCCCCA-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">OCN</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39;-GTGCAGCCTTTGTGTCCAAG-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-GTCAGCCAACTCGTCACAGT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">BSP</td>
			<td rowspan="2">Invitrogen</td>
			<td>forward primer 5&#39; AAAGTGAGAACGGGGAACCT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse primer 5&#39;-GATGCAAAGCCAGAATGGAT-3&#39;</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial ALP primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial CBFA1 primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of mesenchymal stem cells from human alveolar periosteum and effects of vitamin d on osteogenic activity of periosteum-derived cells

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the dental sources of MSCs, the periosteum is an easily accessible tissue, which has been identified to contain MSCs in the cambium layer. However, this source has not yet been widely studied.
Vitamin D3 and 1,25-(OH)2D3 have been demonstrated to stimulate in vitro differentiation of MSCs into osteoblasts. In addition, vitamin C facilitates collagen formation and bone cell growth. However, no study has yet investigated the effects of Vitamin D3 and Vitamin C on MSCs.
Here, we present a method of isolating MSCs from human alveolar periosteum and examine the hypothesis that 1,25-(OH)2D3 may exert an osteoinductive effect on these cells. We also investigate the presence of MSCs in the human alveolar periosteum and assess stem cell adhesion and proliferation. To assess the ability of vitamin C (as a control) and various concentrations of 1,25-(OH)2D3 (10&#8722;10, 10&#8722;9, 10&#8722;8, and 10&#8722;7 M) to alter key mRNA biomarkers in isolated MSCs mRNA expression of alkaline phosphatase (ALP), bone sialoprotein (BSP), core binding factor alpha-1 (CBFA1), collagen-1, and osteocalcin (OCN) are measured using real-time polymerase chain reaction (RT-PCR).

For all quantitative assays, the data are presented as mean &#177; standard deviation (SD). All statistical analyses were performed using Student&#39;s t-test. In total, 34 samples were obtained with a mean participant age of 48.1 &#177; 12.3 y. Eleven of these samples were obtained from male patients and 23 from female patients. Twenty-eight samples were obtained from the molar regions and six from the anterior regions; 26 were obtained from the maxilla and 8 from the mandible. ...

Watch this Scientific Journal Video about Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells at JoVE.com

Isolation of Mesenchymal Stem Cells from Human Alveolar Periosteum and Effects of Vitamin D on Osteogenic Activity of Periosteum-derived Cells

We present a protocol to investigate the mRNA expression biomarkers of periosteum-derived cells (PDCs) induced by vitamin C (vitamin C) and 1,25-dihydroxy vitamin D [1,25-(OH)2D3]. In addition, we evaluate the ability of PDCs to differentiate into osteocytes, chondrocytes, and adipocytes.

Mesenchymal stem cells (MSCs) are present in a variety of tissues and can be differentiated into numerous cell types, including osteoblasts. Among the ...

The overall goal of this study is to investigate the osteoinductive effects of Vitamin D on cultured mesenchymal stem cells that have been harvested from the dental alveolar periosteum. The use of mesenchymal stem cells is a great advance in dentistry for the past decade. They have been successfully isolated and cultured from many part of dental tissue.The objective though is to isolate and culture mesenchymal stem cells from alveolar periosteum and to investigate the osteoinductive potential of Vitamin D compound. Generally speaking, individuals new to this matter will struggle because of the new idea of bone formation in mesenchymal stem cells derived from alveolar periosteum. The laboratory staff demonstration of procedure will be Miss Hsin-Wen Chi.She is a research assistant from my laboratory. To begin, harvest the periosteal tissues from patients during dental surgery using a periosteal separator. Collect slices that are about two by five millimeters.Store the tissue slices in DPBS with antibiotics. At the lab, use a scalpel to thoroughly mince the alveolar periosteal tissue fragments within 24 hours of harvesting. Next, culture the tissue on 35 millimeter petri dishes in the appropriate culturing medium.Use a humidified 37 degree celsius incubator with 5%carbon dioxide gas. Three days later, discard the collected periosteal tissues and change the medium. When the cells have proliferated to about 80%confluence, detach the cultured cells by adding Trypsin.And incubating the culture for three minutes. After this, add 0.8 milliliters of medium to terminate the detachment. After collecting and counting, distribute 5, 000 cells in one milliliter per plate.Mark the plated cells as passage one at this stage. Culture these cells and separate them again after they have proliferated to 80%confluence. The medium with passage one is initially an orange/red color.When the medium begins to turn orange three days later, change it. When the passage one cells reach 80%confluence, create the second to the fifth generation cells by repeating the same procedures used to prepare the zero generation to the first generation. Change the medium twice per week.Four weeks later, assess the potential of the cells to differentiate into an osteogenic lineage by observing the morphology of the cells under a microscope. Stem the cells using a von kossa assay. This assay distinguishes the presence of calcified deposits in your culture.Wash the cells with PBS and the fix them for 30 minutes in one milliliter of 10%Formalin. After this, remove the Formalin and then wash the cells and treat them with 5%silver nitrate. Expose the cells to UV light for one hour.Next, remove the silver nitrate and treat the cells with two milliliters of 5%sodium sulfate four times for three minutes each time. Replace the solution between treatments. Finally, wash the cells twice using distilled water.20 of 34 periosteal tissue samples successfully yielded marrow-derived stem cell colonies. No factors with relation to the donors were found to significantly affect the culturing success. Initially, PDCs formed colonies and spherical clusters with a mix of round and spindle-shaped cells.After the first passage, the cells were homogeneously fibroblast-like with a spindle shape. At the end of the osteogenic differentiation, von kossa assay indicated the possible presence of calcium deposits and osteogenic differentiation. These results strongly indicate that among periosteal cell populations, progenitor cells possess the potential to differentiate into osteogenic cells.The osteogenic precursor cells were also treated with Calcitriol and examined using RTPCR to assess the expression level of alkaline phosphatase which is a strong indicator for early osteogenic differentiation of cells. Both treatment of vitamin C and Calcitriol, an active metabolite of vitamin D, increased alkaline phosphatase expression. Also, collagen one mRNA levels expression increased in response to the same treatments.The expression of bone sialoprotein mRNA was found to increase for the cells treated with a higher concentration of Calcitriol. However, CBFA one, and ostocalcium mRNA expression did not respond to the treatments. After watching this video, the audience should have a good understanding of the mineralization and culture protocol of human alveolar periosteum.Once mastered, this pursuit can be done in four to five weeks if performed properly. When attempting this experiment, it is important to use a careful and sterile technique to avoid contamination of cell.

isolation-mesenchymal-stem-cells-from-human-alveolar-periosteum

Research

JoVE Journal

Bioengineering

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

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 ...

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(&#949;-caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(&#949;-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37&#160;oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged &#8211; the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.

This article describes a range of set-ups for seeding human mesenchymal stem cells onto materials, in this case electrospun yarns, that do not cover the base of standard culture well plates in order to maximize and quantify the number of cells that initially attach compared to the known seeding density.

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell ...

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 ...

Construction and Use of an Electrical Stimulation Chamber for Enhancing Osteogenic Differentiation in Mesenchymal Stem/Stromal Cells In Vitro

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) has been demonstrated to increase MSC osteogenic differentiation in vitro and promote bone healing in clinical settings. Here we describe the construction of an EStim cell culture chamber and its use in treating rat bone-marrow-derived MSC to enhance osteogenic differentiation. We found that treating MSCs with EStim for 7 days results in a significant increase in the osteogenic differentiation, and importantly, this pro-osteogenic effect persists long after (7 days) EStim is discontinued. This approach of pretreating MSCs with EStim to enhance osteogenic differentiation could be used to optimize bone tissue engineering treatment outcomes and, thus, help them to achieve their full therapeutic potential. In addition to this application, this EStim cell culture chamber and protocol can also be used to investigate other EStim-sensitive cell behaviors, such as migration, proliferation, apoptosis, and scaffold attachment.

Here we present a protocol for the construction of a cell culture chamber designed to expose cells to various types of electrical stimulation, and its use in treating mesenchymal stem cells to enhance osteogenic differentiation.

Mesenchymal stem/stromal cells (MSCs) have been used extensively to promote bone healing in tissue engineering approaches. Electrical stimulation (EStim) ...

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 ...

Fibroblast Derived Human Engineered Connective Tissue for Screening Applications

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical stimuli. The current understanding of fibrotic processes, including cardiac fibrosis, remains poor, which hampers the development of new anti-fibrotic therapies. Controllable and reliable human model systems are crucial for a better understanding of fibrosis pathology. This is a highly reproducible and scalable protocol to generate engineered connective tissues (ECT) in a 48-well casting plate to facilitate studies of fibroblasts and the pathophysiology of fibrotic tissue in a 3-dimensional (3D) environment. ECT are generated around the poles with tunable stiffness, allowing for studies under a defined biomechanical load. Under the defined loading conditions, phenotypic adaptations controlled by cell-matrix interactions can be studied. Parallel testing is feasible in the 48-well format with the opportunity for the time-course analysis of multiple parameters, such as tissue compaction and contraction against the load. From these parameters, biomechanical properties such as tissue stiffness and elasticity can be studied.

Presented here is a protocol to generate engineered connective tissues for a parallel culture of 48 tissues in a multi-well plate with double poles, suitable for mechanistic studies, disease modeling, and screening applications. The protocol is compatible with fibroblasts from different organs and species and is exemplified here with human primary cardiac fibroblasts.

Fibroblasts are phenotypically highly dynamic cells, which quickly transdifferentiate into myofibroblasts in response to biochemical and biomechanical ...

Isolation, Expansion, and Differentiation of Mesenchymal Stem Cells from the Infrapatellar Fat Pad of the Goat Stifle Joint

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded during arthroscopic procedures and knee replacement surgeries. Additionally, its removal is associated with minimal donor site morbidity. Recent studies have demonstrated that IFP-MSCs do not lose their proliferation capacity during in vitro expansion and have age-independent osteogenic differentiation potential. IFP-MSCs possess superior chondrogenic differentiation potential compared to bone marrow-derived MSCs (BMSCs) and adipose-derived stem cells (ADSCs). Although these cells are easily obtainable from aged and diseased patients, their effectiveness is limited. Hence, using IFP-MSCs from healthy donors is important to determine their efficacy in biomedical applications. As access to a healthy human donor is challenging, animal models could be a better alternative to enable fundamental understanding. Large animals such as dogs, horses, sheep, and goats play a crucial role in translational research. Amongst these, the goat could be a preferred model since the stifle joint of the goat has the closest anatomy to the human knee joint. Moreover, goat-IFP can fulfill the higher MSC numbers needed for tissue regeneration applications. Furthermore, low cost, availability, and compliance with the 3R principles for animal research make them an attractive model. This study demonstrates a simple protocol for isolating IFP-MSCs from the stifle joint of goats and in vitro culture conditions for their expansion and differentiation. The aseptically isolated IFP from the goat was washed, minced, and digested enzymatically. After filtration and centrifugation, the collected cells were cultured. These cells were adherent, had MSCs-like morphology, and demonstrated remarkable clonogenic ability. Further, they differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating their multipotency. In conclusion, the study demonstrates the isolation and expansion of MSCs, which show potential in tissue engineering and regenerative medicine applications.

Infrapatellar fat pad mesenchymal&#160;stem cells (IFP-MSCs) can be isolated easily from the infrapatellar fat pad of the knee joint. They proliferate well in vitro, form CFU-F colonies, and differentiate into adipogenic, chondrogenic, and osteogenic lineages. Herein, the methodology for the isolation, expansion, and differentiation of IFP-MSCs from goat stifle joint is provided.

The IFP, present in the knee joint, serves as a promising source of MSCs. The IFP is an easily accessible tissue as it is routinely resected and discarded ...

Generation of Human Blood Vessel Organoids from Pluripotent Stem Cells

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study defined aspects of that organ in a tissue culture dish. The breadth and application of human pluripotent stem cell (hPSC)-derived organoid research have advanced significantly to include the brain, retina, tear duct, heart, lung, intestine, pancreas, kidney, and blood vessels, among several other tissues. The development of methods for the generation of human microvessels, specifically, has opened the way for modeling human blood vessel development and disease in vitro and for the testing and analysis of new drugs or tissue tropism in virus infections, including SARS-CoV-2. Complex and lengthy protocols lacking visual guidance hamper the reproducibility of many stem cell-derived organoids. Additionally, the inherent stochasticity of organoid formation processes and self-organization necessitates the generation of optical protocols to advance the understanding of cell fate acquisition and programming. Here, a visually guided protocol is presented for the generation of 3D human blood vessel organoids (BVOs) engineered from hPSCs. Presenting a continuous basement membrane, vascular endothelial cells, and organized articulation with mural cells, BVOs exhibit the functional, morphological, and molecular features of human microvasculature. BVO formation is initiated through aggregate formation, followed by mesoderm and vascular induction. Vascular maturation and network formation are initiated and supported by embedding aggregates in a 3D collagen and solubilized basement membrane matrix. Human vessel networks form within 2-3 weeks and can be further grown in scalable culture systems. Importantly, BVOs transplanted into immunocompromised mice anastomose with the endogenous mouse circulation and specify into functional arteries, veins, and arterioles. The present visually guided protocol will advance human organoid research, particularly in relation to blood vessels in normal development, tissue vascularization, and disease.

This protocol describes the generation of self-organizing blood vessels from human pluripotent and induced pluripotent stem cells. These blood vessel networks exhibit an extensive and connected endothelial network surrounded by pericytes, smooth muscle actin, and a continuous basement membrane.

An organoid is defined as an engineered multicellular in vitro tissue that mimics its corresponding in vivo organ such that it can be used to study ...