This research was supported by the Basic Science Center Program (No. T2288102), the Key Program of the National Natural Science Foundation of China (No. 32230059), the National Natural Science Foundation of China (No. 32301123), the Foundation of Frontiers Science Center for Materiobiology and Dynamic Chemistry (No. JKVD1211002), the Wego Project of Chinese Academy of Sciences (No. (2020) 005), the Project of National Facility for Translational Medicine (Shanghai) (No. TMSK-2021-134), and the China Postdoctoral Science Foundation (No. 2022M721147).

Osteo-Organoid-Derived Cells for Effective HSCT in Irradiated Mice

<ol>
	<li>Copelan, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem-cell+transplantation.">Hematopoietic stem-cell transplantation.</a> New England Journal of Medicine. 354 (17), 1813-1826 (2006).</li><li>Vittayawacharin, P., 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=Autologous+hematopoietic+stem+cell+transplantation+for+a+patient+with+multiple+autoimmune+diseases.">Autologous hematopoietic stem cell transplantation for a patient with multiple autoimmune diseases.</a> American Journal of Hematology. 98 (10), 1659-1662 (2023).</li><li>Xu, L. -. P., 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=Hematopoietic+stem+cell+transplantation+activity+in+China+2019:+a+report+from+the+Chinese+Blood+and+Marrow+Transplantation+Registry+Group.">Hematopoietic stem cell transplantation activity in China 2019: a report from the Chinese Blood and Marrow Transplantation Registry Group.</a> Bone Marrow Transplantation. 56 (12), 2940-2947 (2021).</li><li>Gratwohl, 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=Hematopoietic+stem+cell+transplantation:+a+global+perspective.">Hematopoietic stem cell transplantation: a global perspective.</a> JAMA. 303 (16), 1617-1624 (2010).</li><li>Sakurai, 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=Chemically+defined+cytokine-free+expansion+of+human+haematopoietic+stem+cells.">Chemically defined cytokine-free expansion of human haematopoietic stem cells.</a> Nature. 615 (7950), 127-133 (2023).</li><li>Pineault, N., Abu-Khader, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+umbilical+cord+blood+stem+cell+expansion+and+clinical+translation.">Advances in umbilical cord blood stem cell expansion and clinical translation.</a> Experimental Hematology. 43 (7), 498-513 (2015).</li><li>Liang, G., Liu, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+expansion+of+human+hematopoietic+stem+cells.">Long-term expansion of human hematopoietic stem cells.</a> Cell Regeneration. 12 (1), 18 (2023).</li><li>Wilkinson, A. C., Igarashi, K. J., Nakauchi, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cell+self-renewal+in+vivo+and+ex+vivo.">Haematopoietic stem cell self-renewal in vivo and ex vivo.</a> Nature Reviews Genetics. 21 (9), 541-554 (2020).</li><li>Kumar, S., Geiger, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSC+niche+biology+and+HSC+expansion+ex+vivo.">HSC niche biology and HSC expansion ex vivo.</a> Trends in Molecular Medicine. 23 (9), 799-819 (2017).</li><li>Dai, 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=A+BMP-2-triggered+in+vivo+osteo-organoid+for+cell+therapy.">A BMP-2-triggered in vivo osteo-organoid for cell therapy.</a> Science Advances. 9 (1), 1541 (2023).</li><li>Dai, K., Zhang, W., Deng, S., Wang, J., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sulfated+polysaccharide+regulates+the+homing+of+HSPCs+in+a+BMP-2-triggered+in+vivo+osteo-organoid.">Sulfated polysaccharide regulates the homing of HSPCs in a BMP-2-triggered in vivo osteo-organoid.</a> Advanced Science. 10 (24), e2301592 (2023).</li><li>Alexander, T., Greco, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cell+transplantation+and+cellular+therapies+for+autoimmune+diseases:+overview+and+future+considerations+from+the+Autoimmune+Diseases+Working+Party+(ADWP)+of+the+European+Society+for+Blood+and+Marrow+Transplantation+(EBMT).">Hematopoietic stem cell transplantation and cellular therapies for autoimmune diseases: overview and future considerations from the Autoimmune Diseases Working Party (ADWP) of the European Society for Blood and Marrow Transplantation (EBMT).</a> Bone Marrow Transplantation. 57 (7), 1055-1062 (2022).</li><li>Ding, 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=Infusion+of+haploidentical+HSCs+combined+with+allogenic+MSCs+for+the+treatment+of+ALL+patients.">Infusion of haploidentical HSCs combined with allogenic MSCs for the treatment of ALL patients.</a> Bone Marrow Transplantation. 57 (7), 1086-1094 (2022).</li><li>Sharrack, B., 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=Autologous+haematopoietic+stem+cell+transplantation+and+other+cellular+therapy+in+multiple+sclerosis+and+immune-mediated+neurological+diseases:+updated+guidelines+and+recommendations+from+the+EBMT+Autoimmune+Diseases+Working+Party+(ADWP)+and+the+Joint+Accreditation+Committee+of+EBMT+and+ISCT+(JACIE).">Autologous haematopoietic stem cell transplantation and other cellular therapy in multiple sclerosis and immune-mediated neurological diseases: updated guidelines and recommendations from the EBMT Autoimmune Diseases Working Party (ADWP) and the Joint Accreditation Committee of EBMT and ISCT (JACIE).</a> Bone Marrow Transplantation. 55 (2), 283-306 (2020).</li><li>Nelson, M. R., Roy, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone-marrow+mimicking+biomaterial+niches+for+studying+hematopoietic+stem+and+progenitor+cells.">Bone-marrow mimicking biomaterial niches for studying hematopoietic stem and progenitor cells.</a> J Mater Chem B. 4 (20), 3490-3503 (2016).</li></ol>

The authors declare that they have no competing financial interests.

In this protocol, we present an approach to establish in vivo osteo-organoids with bone marrow-like structures by implanting gelatin sponge scaffolds loaded with BMP-2. We demonstrate that these in vivo osteo-organoids can stably produce therapeutic HSPCs over a long period of time (more than 12 weeks). Compared to existing in vitro expansion or in vivo incubation methods that load cells, this protocol can obtain cell cocktails with diverse cell types, including HSPCs and various immun...

Hematopoietic stem cell transplantation stands as the conventional therapy for a variety of hematological malignancies, as well as numerous inherited and autoimmune disorders1,2,3,4. Nevertheless, the restricted quantity and origin of hematopoietic stem/progenitor cells (HSPCs) have emerged as a substantial impediment to the clinical implementation of hematopoietic stem cell transplantation (HSCT)5,6.
Large-scale in vitro cell expansion is a commonly employed method for harvesting therapeutic cells5,7. Various studies have developed conditions that stimulate HSPCs to self-renew in vitro, typically through the use of a combination of self-renewal agonists (such as cytokines and growth factors) and serum albumin, resulting in the ex vivo expansion of HSPCs8. However, it is important to note that current methods are still time-consuming and challenging to maintain the self-renewal capacity of expanded HSPCs9.
In contrast to the aforementioned method, harvesting cells in vivo presents a new and innovative strategy. This approach involves the establishment of an in vivo osteo-organoid that mimics the native bone marrow structure10,11. To achieve this, we form in vivo osteo-organoids using bone morphogenetic protein-2 (BMP-2)-loaded gelatin scaffolds to obtain abundant and high-quality autologous cell cocktails, including HSPCs. By therapeutically applying these osteo-organoids, we have successfully treated irradiation damage and demonstrated that HSPCs derived from the osteo-organoids can rapidly and stably reconstitute the experimentally impaired immune system.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>AF700-anti-CD11b (M1/70)</td>
			<td>eBioscience</td>
			<td>56-0112-82</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>AF700-anti-Sca-1 (D7)</td>
			<td>BioLegend</td>
			<td>108141</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>APC-anti-CD3e (145-2C11)</td>
			<td>Tonbo</td>
			<td>20-0031-U100</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>bio-anti-CD34 (RAM34)</td>
			<td>eBioscience</td>
			<td>13-0341-82</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>BV421-anti-CD127 (IL-7R&#945;)</td>
			<td>BioLegend</td>
			<td>135023</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>BV510-anti-CD48 (HM48-1)</td>
			<td>BioLegend</td>
			<td>103443</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>BV711-anti-CD16/32 (93)</td>
			<td>BioLegend</td>
			<td>101337</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>Capillary tube&#160;</td>
			<td>Shanghai Huake Labware Co.</td>
			<td>DC616297403604-100mm/0.5mm</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>CORNING</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>GENERAL-REAGENT</td>
			<td>01158566</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediamine tetraacetic acid (EDTA) solution</td>
			<td>Servicebio</td>
			<td>G1105</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid dipotassium salt dihydrate (EDTA-K2)</td>
			<td>Solarbio</td>
			<td>E8651</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC-anti-CD45.2 (104)</td>
			<td>BioLegend</td>
			<td>109806</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>Flowjo</td>
			<td>Becton, Dickinson &#38; Company</td>
			<td></td>
			<td>A flow cytometry.</td>
		</tr>
		<tr>
			<td>Gelatin sponge</td>
			<td>Jiangxi Xiangen Co.</td>
			<td></td>
			<td>Use under sterile conditions.</td>
		</tr>
		<tr>
			<td>HBSS without Ca2+ and Mg2+</td>
			<td>Gibco</td>
			<td>14170112</td>
			<td>HBSS without Ca2+ and Mg2+ can prevent cell aggregation.</td>
		</tr>
		<tr>
			<td>Hematology analyzer</td>
			<td>Sysmex</td>
			<td>pocH-100i Diff</td>
			<td></td>
		</tr>
		<tr>
			<td>iodine swabs</td>
			<td>Xiangtan Mulan Biological Technology Co., Ltd.</td>
			<td>01011</td>
			<td>To prevent the infection after operation.</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>RWD</td>
			<td>R510-22-10&#160;</td>
			<td>To avoid adverse effects of anesthesia waste gases on the environment and laboratory personnel, a gas recovery system should be used in conjunction.</td>
		</tr>
		<tr>
			<td>Kraft paper</td>
			<td></td>
			<td></td>
			<td>absorbent paper</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near IR Dead Cell Staining Kit(used in 3.4.5)</td>
			<td>Thermo Fisher Scientific</td>
			<td>L34962</td>
			<td>A live/dead staining kit. Store at -20 &#176;C. Dissolve in 50 &#956;L of DMSO for working solution.</td>
		</tr>
		<tr>
			<td>lubricating vet ointment</td>
			<td>Pfizer</td>
			<td></td>
			<td>To prevent dryness and counteract the ocular irritations caused by isoflurane.</td>
		</tr>
		<tr>
			<td>Neutral balsam</td>
			<td>Solarbio</td>
			<td>G8590</td>
			<td></td>
		</tr>
		<tr>
			<td>nylon filter</td>
			<td>Shanghai Shangshai Wire Mesh Manufacturing Co., Ltd.</td>
			<td></td>
			<td>Used for cell filtration.</td>
		</tr>
		<tr>
			<td>Paraffin liquid</td>
			<td>Macklin</td>
			<td>P815706</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA) solution</td>
			<td>Servicebio</td>
			<td>G1101</td>
			<td>Immersion fixation is used for routine animal tissues. The volume of fixative used is generally 10-20 times the tissue volume, and fixation at room temperature for 24 hours is sufficient.</td>
		</tr>
		<tr>
			<td>PE-anti-CD45.1 (A20)</td>
			<td>BioLegend</td>
			<td>110708</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>PE-CF594-anti-CD135 (A2F10.1)</td>
			<td>BD Biosciences</td>
			<td>562537</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>PE-Cy5-anti-c-kit (2B8)</td>
			<td>BD Biosciences</td>
			<td>105809</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>PE-Cy7-anti-B220 (RA3-6B2)</td>
			<td>BioLegend</td>
			<td>103222</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>PE-Cy7-anti-CD150 (TC15-12F12.2)</td>
			<td>BioLegend</td>
			<td>115914</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>PE-Dazzle594-anti-CD4 (GK1.5)</td>
			<td>BioLegend</td>
			<td>100456</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>Pentobarbital sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>57-33-0</td>
			<td>Prepare for use at a concentration of 1% (w/v).</td>
		</tr>
		<tr>
			<td>PerCp-Cy5.5-anti-CD8a (53-6.7)</td>
			<td>BioLegend</td>
			<td>100734</td>
			<td>Store at 4 &#176;C. Dilute 1:200 for staining.</td>
		</tr>
		<tr>
			<td>PerCp-Cy5.5-anti-lineage cocktail</td>
			<td>BD Biosciences</td>
			<td>561317</td>
			<td>Store at 4 &#176;C. Dilute 1:10&#160; for staining.</td>
		</tr>
		<tr>
			<td>Red blood cell lysis buffer</td>
			<td>Beyotime</td>
			<td>C3702</td>
			<td>Store at 4 &#176;C. Use in clean bench.</td>
		</tr>
		<tr>
			<td>rhBMP-2</td>
			<td>Shanghai Rebone Biomaterials Co.</td>
			<td></td>
			<td>The concentration of rhBMP-2 in the stock solution is 1.0 mg/mL.</td>
		</tr>
		<tr>
			<td>Staining buffer</td>
			<td>BioLegend</td>
			<td>420201</td>
			<td>Store at 4 &#176;C.</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>GENERAL-REAGENT</td>
			<td>01018114</td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie UV Fixable Viability Kit (used in 6.2.5)</td>
			<td>BioLegend</td>
			<td>423108</td>
			<td>A live/dead staining kit. For reconstitution, bring the kit to room temperature; add 100 &#181;L of DMSO to one vial of Zombie UV dye until fully dissolved.&#160;</td>
		</tr>
	</tbody>
</table>

in vivo osteo-organoid approach for harvesting therapeutic hematopoietic stem/progenitor cells

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids.
We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

As per the protocol, we have created a bioactive scaffold by dripping BMP-2 into a degradable gelatin sponge under sterile conditions. The scaffold was then implanted into the lower limb muscles of mice to establish in vivo osteo-organoids. After an incubation period of 12 weeks, we conducted macroscopic photography, histological analysis, and flow cytometry analysis on the osteo-organoids (Figure 1A). The gelatin sponge was cut into cubes with dimensions of 5 mm x 5 mm x 5 mm, and ...

Read this Scientific Article Protocol about In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells at JoVE.com

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an ...

in-vivo-osteo-organoid-approach-for-harvesting-therapeutic

Research

JoVE Journal

Bioengineering

339 Views.  East China University of Science and Technology. Here, we established in vivo osteo-organoids triggered by bone morphogenetic protein-2-loaded gelatin scaffolds to harvest therapeutic hematopoietic stem/progenitor cells for the reconstruction of a damaged hematopoietic and immune system. Overall, this approach can provide a promising cell source for cell therapies.

Video: In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells

Registered Bioimaging of Nanomaterials for Diagnostic and Therapeutic Monitoring

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end organs. The latter include the lung, RES, and brain and are operative during human immunodeficiency virus type one (HIV-1) infection. Macrophage entry into tissues is notable in areas of active HIV-1 replication and sites of inflammation. In order to assess the potential of macrophages as nanocarriers, superparamagnetic iron-oxide and/or drug laden particles coated with surfactants were parenterally injected into HIV-1 encephalitic mice. This was done to quantitatively assess particle and drug biodistribution. Magnetic resonance imaging (MRI) test results were validated by histological coregistration and enhanced image processing. End organ disease as typified by altered brain histology were assessed by MRI. The demonstration of robust migration of nanoformulations into areas of focal encephalitis provides '"proof of concept" for the use of advanced bioimaging techniques to monitor macrophage migration. Importantly, histopathological aberrations in brain correlate with bioimaging parameters making the general utility of MRI in studies of cell distribution in disease feasible. We posit that using such methods can provide a real time index of disease burden and therapeutic efficacy with translational potential to humans.

Bioimaging methods used to assess cell biodistribution of nanoparticles are applicable for therapeutic and diagnostic monitoring of nanoformulated compounds. The methods described herein are sensitive and specific when assessed by histological coregistration. The methodologies provide a translational pathway from rodent to human applications.

Nanomedications can be carried by blood borne monocyte-macrophages into the reticuloendothelial system (RES; spleen, liver, lymph nodes) and to end ...

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts1-4. More recent studies demonstrate fusion of cells of different types can direct cell fate. The "reversion" or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation5-7. Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease.
Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo8-15. We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system16, 17, we have incorporated a "living" detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo.
To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Programming Stem Cells for Therapeutic Angiogenesis Using Biodegradable Polymeric Nanoparticles

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, heart attack or peripheral arterial diseases. Direct delivery of angiogenic growth factors has the potential to stimulate new blood vessel growth, but is often associated with limitations such as lack of targeting and short half-life in vivo. Gene therapy offers an alternative approach by delivering genes encoding angiogenic factors, but often requires using virus, and is limited by safety concerns. Here we describe a recently developed strategy for stimulating vascular growth by programming stem cells to overexpress angiogenic factors in situ using biodegradable polymeric nanoparticles. Specifically our strategy utilized stem cells as delivery vehicles by taking advantage of their ability to migrate toward ischemic tissues in vivo. Using the optimized polymeric vectors, adipose-derived stem cells were modified to overexpress an angiogenic gene encoding vascular endothelial growth factor (VEGF). We described the processes for polymer synthesis, nanoparticle formation, transfecting stem cells in vitro, as well as methods for validating the efficacy of VEGF-expressing stem cells for promoting angiogenesis in a murine hindlimb ischemia model.

We describe the method of programming stem cells to overexpress therapeutic factors for angiogenesis using biodegradable polymeric nanoparticles. Processes described include polymer synthesis, transfecting adipose-derived stem cells in vitro, and validating the efficacy of programmed stem cells to promote angiogenesis in a murine hindlimb ischemia model.

Controlled vascular growth is critical for successful tissue regeneration and wound healing, as well as for treating ischemic diseases such as stroke, ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

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 ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Rigid Embedding of Fixed and Stained, Whole, Millimeter-Scale Specimens for Section-free 3D Histology by Micro-Computed Tomography

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in every tissue to be identified and characterized. Our laboratory is actively working to make technological advances in X-ray micro-computed tomography (micro-CT) that will bring the diagnostic power of histology to the study of full tissue volumes at cellular resolution (i.e., an X-ray Histo-tomography modality). Toward this end, we have made targeted improvements to the sample preparation pipeline. One key optimization, and the focus of the present work, is a straightforward method for rigid embedding of fixed and stained millimeter-scale samples. Many of the published methods for sample immobilization and correlative micro-CT imaging rely on placing the samples in paraffin wax, agarose, or liquids such as alcohol. Our approach extends this work with custom procedures and the design of a 3-dimensional printable apparatus to embed the samples in an acrylic resin directly into polyimide tubing, which is relatively transparent to X-rays. Herein, sample preparation procedures are described for the samples from 0.5 to 10 mm in diameter, which would be suitable for whole zebrafish larvae and juveniles, or other animals and tissue samples of similar dimensions. As proof of concept, we have embedded the specimens from Danio, Drosophila, Daphnia, and a mouse embryo; representative images from 3-dimensional scans for three of these samples are shown. Importantly, our methodology leads to multiple benefits including rigid immobilization, long-term preservation of laboriously-created resources, and the ability to re-interrogate samples.

We developed protocols and designed a custom apparatus to enable embedding of millimeter-scale specimens. We present sample preparation procedures with an emphasis on embedding in acrylic resin and polyimide tubing to achieve rigid immobilization and long-term storage of specimens for the interrogation of tissue architecture and cell morphology by micro-CT.

For over a hundred years, the histological study of tissues has been the gold standard for medical diagnosis because histology allows all cell types in ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...