Name: prospective, randomized, and controlled study of a human umbilical cord mesenchymal stem cell injection for treating diabetic foot ulcers
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Description: Watch this Scientific Journal Video about Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers at JoVE.com

The authors thank the Affiliated Hospital of Xuzhou Medical University for its cooperation, including the recruitment and follow-up of patients with diabetic foot wounds. The authors also thank the patients who participated in the patient needs survey during the design of this study.
The author(s) announce the receipt of the following financial support for research, authorship, and/or publication of this article: National Natural Science Foundation of China 82172224, Postgraduate Research &#38; Practice Innovation Program of Jiangsu Province (SJCX22-1271), and&#160;the Innovation &#38; Technology Commission (Health@InnoHK).

This prospective, single-center, randomized, controlled clinical study was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (XYFY2021-KL124-02). The study started in July 2021 and will continue until July 2023; 60 patients were recruited in this experiment. All patients signed an informed consent that allowed the researchers to use their clinical materials and biological data.
1. Recruitment of patients
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	<li>Inclusion criteria:
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<ol>
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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pathway+to+foot+ulceration+in+diabetes.">The pathway to foot ulceration in diabetes.</a> The Medical Clinics of North America. 97 (5), 7755-7790 (2013).</li><li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+prevalence+of+foot+ulceration+and+its+correlates+in+type+2+diabetic+patients:+a+population-based+study.">The prevalence of foot ulceration and its correlates in type 2 diabetic patients: a population-based study.</a> Diabetic Medicine. 11 (5), 480-484 (1994).</li><li>Margolis, D. J., Jeffcoate, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+of+foot+ulceration+and+amputation:+Can+global+variation+be+explained.">Epidemiology of foot ulceration and amputation: Can global variation be explained.</a> The Medical Clinics of North America. 97 (5), 791-805 (2013).</li><li>Burgess, J. L., Wyant, W. A., Abdo Abujamra, B., Kirsner, R. S., Jozic, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diabetic+wound-healing+science.">Diabetic wound-healing science.</a> Medicina. 57 (10), 1072 (2021).</li><li>Behr, B., Ko, S. H., Wong, V. W., Gurtner, G. C., Longaker, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells.">Stem cells.</a> Plastic and Reconstructive Surgery. 126 (4), 1163-1171 (2010).</li><li>Moon, K. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potential+of+allogeneic+adipose-derived+stem+cell-hydrogel+complex+for+treating+diabetic+foot+ulcers.">Potential of allogeneic adipose-derived stem cell-hydrogel complex for treating diabetic foot ulcers.</a> Diabetes. 68 (4), 837-846 (2019).</li><li>Bacakova, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells:+Their+source,+potency+and+use+in+regenerative+therapies+with+focus+on+adipose-derived+stem+cells+-+A+review.">Stem cells: Their source, potency and use in regenerative therapies with focus on adipose-derived stem cells - A review.</a> Biotechnology Advances. 36 (4), 1111-1126 (2018).</li><li>Dulchavsky, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=marrow-derived+stromal+cells+(BMSCs)+interact+with+fibroblasts+in+accelerating+wound+healing.">marrow-derived stromal cells (BMSCs) interact with fibroblasts in accelerating wound healing.</a> Journal of Investigative Surgery. 21 (5), 270-279 (2008).</li><li>Hu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+derived+from+pioglitazone-pretreated+MSCs+accelerate+diabetic+wound+healing+through+enhancing+angiogenesis.">Exosomes derived from pioglitazone-pretreated MSCs accelerate diabetic wound healing through enhancing angiogenesis.</a> Journal of Nanobiotechnology. 19 (1), 150 (2021).</li><li>Meng, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CircARHGAP12+triggers+mesenchymal+stromal+cell+autophagy+to+facilitate+its+effect+on+repairing+diabetic+wounds+by+sponging+miR-301b-3p/ATG16L1+and+miR-301b-3p/ULK2.">CircARHGAP12 triggers mesenchymal stromal cell autophagy to facilitate its effect on repairing diabetic wounds by sponging miR-301b-3p/ATG16L1 and miR-301b-3p/ULK2.</a> The Journal of Investigative Dermatology. 142 (7), 1976.e4-1989.e4 (2022).</li><li>Shen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomal+microRNA+rectangle93+rectangle3p+secreted+by+bone+marrow+mesenchymal+stem+cells+downregulates+apoptotic+peptidase+activating+factor+1+to+promote+wound+healing.">Exosomal microRNA rectangle93 rectangle3p secreted by bone marrow mesenchymal stem cells downregulates apoptotic peptidase activating factor 1 to promote wound healing.</a> Bioengineered. 13 (1), 27-37 (2022).</li><li>Alberti, K. G., Zimmet, P. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definition,+diagnosis+and+classification+of+diabetes+mellitus+and+its+complications.+Part+1:+Diagnosis+and+classification+of+diabetes+mellitus+provisional+report+of+a+WHO+consultation.">Definition, diagnosis and classification of diabetes mellitus and its complications. Part 1: Diagnosis and classification of diabetes mellitus provisional report of a WHO consultation.</a> Diabetic Medicine. 15 (7), 539-553 (1998).</li><li>Lijmer, J. G., Hunink, M. G., vanden Dungen, J. J., Loonstra, J., Smit, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ROC+analysis+of+noninvasive+tests+for+peripheral+arterial+disease.">ROC analysis of noninvasive tests for peripheral arterial disease.</a> Ultrasound in Medicine &amp; Biology. 22 (4), 391-398 (1996).</li><li>Wagner Jr, F. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dysvascular+foot:+A+system+for+diagnosis+and+treatment.">The dysvascular foot: A system for diagnosis and treatment.</a> Foot &amp; Ankle. 2 (2), 64-122 (1981).</li><li>Griffin, J. W., Tolley, E. A., Tooms, R. E., Reyes, R. A., Clifft, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+photographic+and+transparency-based+methods+for+measuring+wound+surface+area.">A comparison of photographic and transparency-based methods for measuring wound surface area.</a> Physical Therapy. 73 (2), 117-122 (1993).</li><li>Lipsky, B. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+and+treatment+of+diabetic+foot+infections.">Diagnosis and treatment of diabetic foot infections.</a> Plastic and Reconstructive Surgery. 117 (7 Suppl), 212S-238S (2006).</li><li>Huang, Y. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+a+novel+macrophage-regulating+drug+on+wound+healing+in+patients+with+diabetic+foot+ulcers:+A+randomized+clinical+trial.">Effect of a novel macrophage-regulating drug on wound healing in patients with diabetic foot ulcers: A randomized clinical trial.</a> JAMA Network Open. 4 (9), e2122607 (2021).</li><li>Yarahmadi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+platelet-rich+plasma-fibrin+glue+dressing+in+combination+with+oral+vitamin+E+and+C+for+treatment+of+non-healing+diabetic+foot+ulcers:+A+randomized,+double-blind,+parallel-group,+clinical+trial.">The effect of platelet-rich plasma-fibrin glue dressing in combination with oral vitamin E and C for treatment of non-healing diabetic foot ulcers: A randomized, double-blind, parallel-group, clinical trial.</a> Expert Opinion on Biological Therapy. 21 (5), 687-696 (2021).</li><li>Virador, G. M., de Marcos, L., Virador, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+wound+healing:+Refractory+wounds+and+novel+solutions.">Skin wound healing: Refractory wounds and novel solutions.</a> Methods in Molecular Biology. 1879, 221-241 (2019).</li><li>Um, S., Ha, J., Choi, S. J., Oh, W., Jin, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospects+for+the+therapeutic+development+of+umbilical+cord+blood-derived+mesenchymal+stem+cells.">Prospects for the therapeutic development of umbilical cord blood-derived mesenchymal stem cells.</a> World Journal of Stem Cells. 12 (12), 1511-1528 (2020).</li><li>Xiao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+biological+characteristics+of+human+bone+marrow+hematopoietic+stem+cell+senescence.">Dynamic biological characteristics of human bone marrow hematopoietic stem cell senescence.</a> Scientific Reports. 12, 17071 (2022).</li><li>Zhang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Safety+and+immunological+responses+to+human+mesenchymal+stem+cell+therapy+in+difficult-to-treat+HIV-1-infected+patients.">Safety and immunological responses to human mesenchymal stem cell therapy in difficult-to-treat HIV-1-infected patients.</a> AIDS. 27 (8), 1283-1293 (2013).</li><li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+umbilical+cord+mesenchymal+stem+cell+promotes+angiogenesis+via+integrin+beta1/ERK1/2/HIF-1alpha/VEGF-A+signaling+pathway+for+off-the-shelf+breast+tissue+engineering.">Human umbilical cord mesenchymal stem cell promotes angiogenesis via integrin beta1/ERK1/2/HIF-1alpha/VEGF-A signaling pathway for off-the-shelf breast tissue engineering.</a> Stem Cell Research &amp; Therapy. 13 (1), 99 (2022).</li><li>Li, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-inflammatory+and+immunomodulatory+effects+of+the+extracellular+vesicles+derived+from+human+umbilical+cord+mesenchymal+stem+cells+on+osteoarthritis+via+M2+macrophages.">Anti-inflammatory and immunomodulatory effects of the extracellular vesicles derived from human umbilical cord mesenchymal stem cells on osteoarthritis via M2 macrophages.</a> Journal of Nanobiotechnology. 20 (1), 38 (2022).</li><li>Qin, H. L., Zhu, X. H., Zhang, B., Zhou, L., Wang, W. 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DFUs are a major global public health problem and a key cause of lower limb amputations and poor health-related quality of life19,20. At present, clinical management is still dominated by conventional debridement, hyperbaric oxygen therapy, vacuum sealing drainage (VSD), and conservative management. Larger wounds often require the transplantation of skin and skin flaps. Many patients suffer from long-term and repeated illnesses that cause severe physical and ment...

Diabetes mellitus is a disease that affects individuals worldwide, and the World Health Organization (WHO) predicts that the number of people with diabetes mellitus will increase from 285 million in 2010 to 439 million in 20301. Diabetic foot ulcers (DFUs) are one of the most serious complications of diabetes and are main contributors to non-traumatic lower-extremity amputations across the world2,3,4,5.
Recently, stem cells have flourished as a therapy due to their pluripotency, self-renewal, and ability to promote the secretion of regenerative cytokines6,7. A previous clinical experiment showed that fat-derived stem cell gels had a positive effect on the treatment of foot ulcers in chronic diabetes8. The authors verified the effectiveness of using stem cells to treat diabetic wounds in 59 patients. At week 12, the rate of complete wound closure in the treatment and control groups was 82% and 53%, respectively, which indicates that stem cells are effective for the treatment of refractory diabetic wounds. Overall, the ability of stem cells to regenerate, replace, repair, and differentiate has given infinite hope to the life science community9.
In 2008, Dulchavsky et al.10 used grafts containing autologous bone marrow stromal cells (BMSCs) to treat 20 cases of non-healing wounds of different causes. Histological examinations showed that the skin wounds of 18 patients were completely re-epithelialized. Similarly, the use of allogeneic non-diabetic mesenchymal stem cells (MSCs) in gelatin scaffolds for the treatment of nonunion diabetic wounds can promote angiogenesis, increase re-epithelialization, and reduce the ulcer area11. However, there are few cases of stem cell treatment of diabetic foot wounds in domestic and foreign clinical research studies; most are only case reports or exploratory clinical research lacking a strict experimental design, and there are few large samples with good design or randomized controlled clinical trials. As stem cells are not common drugs or biological products, the preparation methods and quality controls differ between studies. Data from one study may not fully reflect the safety of all stem cells from the same species. Therefore, we further summarized the relevant basic research and preclinical experiments with human umbilical cord mesenchymal stem cells (HUCMSCs) and systematically evaluated the safety and efficacy of their clinical application. On this basis, the injection of mesenchymal stem cells from the human umbilical cord to repair diabetic foot wounds was developed as a treatment. This study aims to verify the efficacy and safety of stem cell repair of diabetic foot wounds in clinical use.
In summary, stem cell therapy has a broad spectrum of applications and great potential and represents a promising new medical treatment method. With the continued support of the National Natural Science Foundation of China (No. 81571901, No. 81501671, and No. 82172224), we conducted a series of studies on the treatment of diabetic wounds with HUCMSCs. We have published more than 20 related articles in the Journal of Investigative Dermatology, Stem Cell Research and Therapy, and Cell Death and Disease and have applied for three national invention patents, thus accumulating a large foundation of research12,13. Here, we provide a standard approach to evaluate the injection of HUCMSCs for diabetic foot wound repair. This standard procedure has been approved by Chinese drug clinical trials (Trial registration number: Chinese drug clinical trials: MR-32-21-015759, [Initial Release: 10/20/2021]).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Silver Iin Wound Dressing</td>
			<td>Shandong Cheerain Medical Co.,Ltd.&#160;</td>
			<td>20152640521</td>
			<td>Sterile silver ion dressing for medical use (Type F) 10 cm x 10 cm</td>
		</tr>
		<tr>
			<td>Human Umbilical Cord Mesenchymal Stem Cells Injection</td>
			<td>Shandong Qilu Cell Therapy Engineering Technology Co., Ltd.</td>
			<td>32183185-X</td>
			<td>Main components: human umbilical cord mesenchymal stem cells. Pharmacological effect: non-specific immunomodulator can enhance the secretion of growth factor, vasoactive factor and anti-inflammatory factor, improve the anti infection ability of human body, eliminate inflammation, promote angiogenesis and ulcer healing.</td>
		</tr>
		<tr>
			<td>Sterile mesh film transparent dressing</td>
			<td>Smith &#38; Nephew</td>
			<td>20162644490</td>
			<td>Sterile mesh film transparent dressing (used for wound area measurement) 6 cm x 7 cm</td>
		</tr>
	</tbody>
</table>

prospective, randomized, and controlled study of a human umbilical cord mesenchymal stem cell injection for treating diabetic foot ulcers

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with dressing changes, hyperbaric oxygen, and vacuum sealing drainage are the main conservative treatments in clinical practice, and large wounds often require skin grafts or skin flap grafts. However, the treatment effects are not ideal, and many complications exist. Due to its complex pathogenesis, long treatment time, significant associated difficulties, and high disability rate, diabetic foot ulcers cause a heavy burden to patients, society, and medical care. According to our previous study, the pharmacological effects of human umbilical cord blood stem cells include nonspecific immune regulation; increased secretion of growth factors, vasoactive factors, and anti-inflammatory factors; enhanced anti-infectious ability of the human body; elimination of inflammation; and promotion of angiogenesis and ulcer healing. These effects suggest stem cells may be useful as an autologous or allogeneic treatment for refractory wounds. Therefore, we are conducting a clinical trial to treat refractory diabetic wounds with human umbilical cord stem cells in our clinic for diabetic foot ulcer patients who meet the inclusion criteria.

At present, our research is still in the patient recruitment phase, and we have now completed three patients in the HUCMSCs treatment group and three patients in the control group with silver ion dressings, giving a total of six patients with chronic diabetic foot wounds. The average size of the ulcer area of a patient in the HUCMSCs treatment group was 3.5 cm2, and this was reduced to 2.6 cm2, 1.8 cm2, and 1.25 cm2 on the 8th, 15th, and 22nd days after treatment with HUCMSCs (...

Watch this Scientific Journal Video about Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers at JoVE.com

Prospective, Randomized, and Controlled Study of a Human Umbilical Cord Mesenchymal Stem Cell Injection for Treating Diabetic Foot Ulcers

The present protocol describes a prospective, randomized, controlled clinical study that evaluates a human umbilical cord mesenchymal stem cell injection for the treatment of chronic diabetic foot ulcers.

With the development of society and the economy, the incidence of diabetic foot ulcers continues to increase. Currently, conventional debridement with ...

Our protocol aim to provide a uniform model for system cells injection to treat diabetic ulcers to evaluate the safety and effectiveness of this treatment. We provide a research standard for stem cells for treating diabetic ulcer, which has the advantages of the high consistencies and the repeatability. Diabetic ulcer is just one of the representative of common refractory ulcer.We hope to apply stem cells to the treatment of other ulcers or other diseases through this protocol. When using this method to treat the body ulcers, it is necessary to ensure the safety of the source of stem cells and to monitor whether patients experience immune rejection. Through visual demonstration, we hope to provide standard and references for other researchers in the treatment of diabetic ulcers or other refractory wounds.Begin by disinfecting the wound site with povidone iodine solution. Remove any necrotic tissue using surgical scissors. Now use the syringe to aspirate 50 milliliters of saline to clean the wound surface.Measure the wound area using the sterile foil edge hook method. Apply the sterile film transparent dressing to the wound surface. Outline the shape of the wound surface with a marker to calculate the wound area.In the stem cell therapy group, inject the human umbilical cord mesenchymal stem cells into the wound periphery and base. Cover the wound surface with an appropriately sized sterile dressing and then bandage it. In the conventional wound treatment group, once the site has been cleaned, measured, and marked as described earlier, cover the wound with a silver ion dressing before bandaging it.The wounds treated with the human umbilical cord mesenchymal stem cells reduced in size from 3.5 square centimeters to 2.6 square centimeters on the eighth day. By the 15th day, the wound size was 1.8 square centimeters. It was further reduced to 1.25 square centimeters on the 22nd day post-treatment.The wounds treated with a conventional silver ion dressing also decreased in size from 1.25 square centimeters to 0.875 square centimeters on the eighth day. By the 15th day, the wound size was 0.8 square centimeters. A slight reduction to 0.75 square centimeters was seen by the 22nd day.The critical step of this experiment is to inject stem cells into the wound at a fixed point and a fixed time. Moreover, long-term followup is required for laboratory tests. According to the biological characteristics of stem cells, following this procedure, it can be applied to burn wounds, infection wounds, and et cetera, which plays an important role in the research of wound healing.After the development of stem cells technology for treating diabetic ulcers, we believe it can play a huge role in most wound treatment through its ability to secrete growth factors and vasoactive factors effectively.

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Research

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Bioengineering

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 ...

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 ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Mouse Model of Pressure Ulcers After Spinal Cord Injury

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony prominences. There is, however, little known about the impact of SCI on skin wound healing in the context of animal models in controlled experimental settings. In this study, a simple, non-invasive, reproducible and clinically relevant mouse model of PUs in the context of complete SCI is presented. Adult male mice (Balb/c, 10 weeks old) were shaved and depilated. Post-depilation (24 h), mice were subjected to laminectomy followed by complete spinal cord transection (T9-T10 vertebrae). Immediately after, a skin fold on the back of the mice was lifted and sandwiched between two magnetic discs held in place for next 12 h, thus creating an ischemic area that developed into a PU over the following days. The wounded areas demonstrated tissue edema and epidermal disappearance by day 3 post-magnet application. PUs spontaneously developed and healed. Healing was, however, slower in the SCI mice compared to control non-SCI mice when the wound was created below the level of SCI. Conversely, no difference in healing was seen between SCI and control non-SCI mice when the wound was created above the level of SCI. This model is a potentially useful tool to study the dynamics of skin PU development and healing after SCI, as well as to test therapeutic approaches that may help heal such wounds.

Here, we describe a simple method to induce clinically relevant skin pressure ulcers (PUs) in a mouse model of spinal cord injury (SCI). This model can be used in pre-clinical studies to screen for different therapeutics for healing PUs in SCI patients.

Pressure ulcers (PUs) are common debilitating complications of traumatic spinal cord injury (SCI) and tend to occur in soft tissues around bony ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

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 ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...

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, ...

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...