The protocol for the generation of human CD40-activated B cells from PBMC is divided into two parts: Part A demonstrates the preparation of CD40 ligand expressing NIH/3T3 cells, which will be used as plate-bound feeder cells. Part B describes the actual CD40-B culture.
A. Preparation of feeder cells (tCD40L NIH/3T3)
The tCD40L NIH/3T3 is an adherent murine fibroblast cell line, which should never become completely confluent. The cells are therefore splitted twice per week. Culturing over more than 6 weeks is not recommended.
<ol>
 <li>Remove old medium from the primary culture with a sterile pipette and wash cells with 10 mL of 1x PBS. Aspirate the PBS after washing.</li>
 <li>Add 4 mL Trypsin/EDTA in a 75 cm2 flask for 5-10 minutes at 37&deg;C. Use gentle tapping to detach the cells.</li>
 <li>Add 10 mL of wild type medium and swivel gently.</li>
 <li>Transfer the cell suspension into a 50 mL tube with a sterile pipette and spin the cells down at 225 x g for 5 min.</li>
 <li>Remove the supernatant and resuspend the pellet in 10 mL of wild type medium. Count the cell number of an aliquot of the cell suspension and prepare three 50 mL tubes with the appropriate number of cells:
 <ol>
 <li>1.5 x 106 cells for subculturing</li>
 <li>0.2 x 106 cells/well for irradiation used for the CD40-B cell culture</li>
 <li>remainder to freeze (if needed).</li>
 </ol>
 </li>
 <li>Spin the cells down at 225 x g for 5 min.</li>
 <li>Remove the supernatant.
 <ol>
 <li>For subculturing: resuspend 1.5 x 106 cells in 10 mL wild type medium in a 75 cm2 cell culture flask (cell density 1.5 x 105 cells/mL), add G-418 [0.7 mg/mL] and incubate the cells at 37&deg;C with 5 % CO2. Split the cells twice per week.</li>
 <li>For the CD40-B cell culture: You need 1.2 x 106 cells for one 6-well plate. Resuspend the cells in wild type medium at a density of 0.1 x 106 cells/mL and irradiate them at 78 Gy. Plate 2 mL of the cell suspension into each well and incubate them at 37&deg;C with 5 % CO2. Use this prepared plates for B-cell stimulation when tCD40L NIH/3T3 cells are adherent (at least 4 hours: check adherence with the microscope; do not wait more than 24 h to start B-cell stimulation). (Continue with B.)</li>
 </ol>
 </li>
</ol>
B. CD40-B cell culture
I. Preparation of PBMCs for CD40-stimulation (day 0):
Please note: before you continue ascertain that feeder cells are adherent. Always add fresh solutions of interleukin-4 and cyclosporin A to the growth medium immediately before use.
<ol>
 <li>Take PBMCs, either fresh or appropriately thawed. Resuspend PBMCs twice in 50mL of 1x PBS to wash them and spin down first time at 265 x g for 7 min and a second time at 190 x g for 7 min to remove other cells. Discard supernatant and resuspend the cells in 20 mL of PBS. Determine the cell number in an aliquot of the cell suspension.</li>
 <li>Spin down required amount of cells at 225 x g for 5 min. For a 6-well plate 4 x 106 cells / well are needed, thus 24 x 106 cells per plate.</li>
 <li>Remove the supernatant and resuspend the PBMC at 1 x 106 cells/mL in CD40-B culture medium freshly supplemented with 50 U/mL of interleukin-4 as a growth factor and 0.63 &mu;g/mL cyclosporine A to prevent outgrowth of T-cells (Given concentrations refer to one mL culture medium!).</li>
 <li>Remove the supernatant from 6-well plate pre-incubated with tCD40L NIH/3T3 cells.</li>
 <li>Wash the adherent tCD40L NIH/3T3 cells with 2 mL PBS per well first and in a second step with 2 mL of CD40-B washing medium.</li>
 <li>Gently add 4 mL of PBMC suspension (1 x 106 cells/mL) to each well of the 6-well plate.</li>
 <li>Incubate the cells at 37&deg;C with 5 % CO2.</li>
 <li>On day 7, reculture the cells (Continue with 3.2.).</li>
</ol>
II. Recultivation of CD40-B cells (day 7 and then every 3-4 days):
<ol>
 <li>Harvest clusters of CD40-B cells from 6-well plate by resuspending with a 10 mL pipette and pool them into a 50 mL tube.</li>
 <li>Spin down at 225 x g for 7 minutes and completely replace the supernatant with CD40-B washing medium. While counting the cell amount of an aliquot, spin the cells down at 225 x g for 5 minutes. Resuspend CD40-B cells in CD40-B culture medium at a concentration of 1 x 106 cells/mL.</li>
 <li>Add fresh solutions of interleukin-4 in a concentration of 50 U/mL and 0.63 &mu;g/mL cyclosporine A to the medium.</li>
 <li>Remove the supernatant from a 6-well plate pre-incubated with tCD40L NIH/3T3 cells.</li>
 <li>Wash the adherent cells with 2 mL PBS per well first and in a second step with 2 mL of CD40-B washing medium.</li>
 <li>Gently add 4 mL (1 x 106 cells/mL) of CD40-B suspension to each well of the 6-well plate.</li>
 <li>Incubate plates at 37&deg;C with 5 % CO2.</li>
 <li>Subculture cells again every 3-4 days to end up with highly pure human CD40-activated B cells after a total of 14 days.</li>
</ol>
C. Trouble-shooting &#x2013; What if CD40-B cells do not grow?
<ol>
 <li>Have you checked for CD40 ligand expression of feeder cells?</li>
 <li>The plate-bound feeder cells used for the stimulation should not be older than 24h?</li>
 <li>Is a contamination with mycoplasma possible?</li>
 <li>Was the interleukin-4 solution used for supplementation freshly thawed and had the appropriate biological activity?</li>
 <li>Was cyclosporin A added in the correct concentration?</li>
</ol>
<img src="/files/ftp_upload/1373/1373fig1tmb.jpg" border="0" alt="Figure 1" /> Figure 1. Please <a href="https://www.jove.com/files/ftp_upload/1373/1373fig1.jpg">click here</a> for a larger version of figure 1.
<img src="/files/ftp_upload/1373/1373fig2tmb.jpg" border="0" alt="Figure 2" /> Figure 2. Please <a href="https://www.jove.com/files/ftp_upload/1373/1373fig2.jpg">click here</a> for a larger version of figure 2.

<ol>
	<li>Schultze, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+human+B+cells:+an+alternative+source+of+highly+efficient+antigen+presenting+cells+to+generate+autologous+antigen-specific+T+cells+for+adoptive+immunotherapy.">CD40-activated human B cells: an alternative source of highly efficient antigen presenting cells to generate autologous antigen-specific T cells for adoptive immunotherapy.</a> J Clin Invest. 100, 2757-2765 (1997).</li><li>Schultze, J. L., Grabbe, S., vonBergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DCs+and+CD40-activated+B+cells:+current+and+future+avenues+to+cellular+cancer+immunotherapy.">DCs and CD40-activated B cells: current and future avenues to cellular cancer immunotherapy.</a> Trends Immunol. 25, 659-664 (2004).</li><li>Bergwelt-Baildon, M. v. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+express+full+lymph+node+homing+triad+and+induce+T-cell+chemotaxis:+potential+as+cellular+adjuvants.">CD40-activated B cells express full lymph node homing triad and induce T-cell chemotaxis: potential as cellular adjuvants.</a> Blood. 107, 2786-2789 (2006).</li><li>Wiesner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+immortalization+of+human+B+cells+by+CD40+ligation.">Conditional immortalization of human B cells by CD40 ligation.</a> PLoS ONE. 3, 1464-14 (2008).</li><li>Urashima, M., Chauhan, D., Uchiyama, H., Freeman, G. J., Anderson, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40+ligand+triggered+interleukin-6+secretion+in+multiple+myeloma.">CD40 ligand triggered interleukin-6 secretion in multiple myeloma.</a> Blood. 85, 1903-1912 (1995).</li><li>Lapointe, R., Bellemare-Pelletier, A., Housseau, F., Thibodeau, J., Hwu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-stimulated+B+lymphocytes+pulsed+with+tumor+antigens+are+effective+antigen-presenting+cells+that+can+generate+specific+T+cells.">CD40-stimulated B lymphocytes pulsed with tumor antigens are effective antigen-presenting cells that can generate specific T cells.</a> Cancer Res. 63, 2836-2843 (2003).</li><li>von Bergwelt-Baildon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+primary+and+memory+cytotoxic+T+lymphocyte+responses+are+efficiently+induced+by+means+of+CD40-activated+B+cells+as+antigen-presenting+cells:+potential+for+clinical+application.">Human primary and memory cytotoxic T lymphocyte responses are efficiently induced by means of CD40-activated B cells as antigen-presenting cells: potential for clinical application.</a> Blood. 99, 3319-3325 (2002).</li><li>Kondo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CD40-activated+B+cells+can+be+generated+in+high+number+and+purity+in+cancer+patients:+analysis+of+immunogenicity+and+homing+potential.">CD40-activated B cells can be generated in high number and purity in cancer patients: analysis of immunogenicity and homing potential.</a> Clin Exp Immunol. 155, 249-256 (2009).</li><li>Mason, N. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-loaded+CD40-activated+B+cells+stimulate+antigen-specific+T-cell+responses+in+dogs+with+spontaneous+lymphoma.">RNA-loaded CD40-activated B cells stimulate antigen-specific T-cell responses in dogs with spontaneous lymphoma.</a> Gene Ther. 15, 955-965 (2008).</li></ol>

A. Preparation of Media:
<table border="1"cellspacing="0">
<tbody>
 <tr>
 <td colspan="2">Feeder cell wild type medium</td>
 <td colspan="2">Feeder cell selection medium</td>
 </tr>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr>
 <td>DMEM-Ham's / F12</td>
 <td>&nbsp;</td>
 <td>DMEM-Ham's / F12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td> L-Glutamine</td>
 <td>365 &mu;g/mL</td>
 <td> L-Glutamine</td>
 <td>365 &mu;g/mL</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>10 %</td>
 <td>FBS</td>
 <td>10 %</td>
 </tr>
 <tr>
 <td>HEPES</td>
 <td>10 mM</td>
 <td>HEPES</td>
 <td>10 mM</td>
 </tr>
 <tr>
 <td>Gentamycin</td>
 <td>15 &mu;g/mL</td>
 <td>Gentamycin</td>
 <td>15 &mu;g/mL</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>G-418</td>
 <td>0.7 mg/mL</td>
 </tr>
</table>
<table cellspacing="0" border="1">
 <tbody>
 <tr>
 <td colspan="2">CD40-B washing medium</td>
 <td colspan="2">CD40-B culture medium</td>
 </tr>
 <tr>
 <td>Reagent</td>
 <td>Concentration</td>
 <td>Reagent</td>
 <td>Concentration</td>
 </tr>
 <tr></tr>
 </tbody>
 <tbody>
 <tr>
 <td>IMDM</td>
 <td> 
 </td>
 <td>IMDM</td>
 <td> 
 </td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td>L-Glutamine</td>
 <td>584 	&mu;g/mL</td>
 <td>L-Glutamine</td>
 <td>584 	&mu;g/mL</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td>HEPES</td>
 <td>25 	mM</td>
 <td>HEPES</td>
 <td>25 	mM</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td>Gentamycin</td>
 <td>15 	&mu;g/mL</td>
 <td>Gentamycin</td>
 <td>15 	&mu;g/mL</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td> 
 </td>
 <td> 
 </td>
 <td>rh 	Transferrin</td>
 <td>50 	&mu;g/mL</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td> 
 </td>
 <td> 
 </td>
 <td>rh 	Insulin</td>
 <td>5 	&mu;g/mL</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td> 
 </td>
 <td> 
 </td>
 <td>AB-Humanserum </td>
 <td>10 	%</td>
 </tr>
 </tbody>
</table>
B. Miscellaneous Reagents:
<table cellspacing="0" border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Order No</td>
 </tr>
 <tr></tr>
 <tr>
 <td>DMEM / Ham's F12</td>
 <td>PAA</td>
 <td>Cat No: E15-813</td>
 </tr>
 <tr>
 <td>IMDM</td>
 <td>Invitrogen Gibco&reg;</td>
 <td>REF 21980-032</td>
 </tr>
 <tr>
 <td>Dulbecco's PBS (10x)</td>
 <td>PAA</td>
 <td>Cat No H15-011</td>
 </tr>
 <tr>
 <td>AB-Human Serum</td>
 <td>Invitrogen</td>
 <td>Cat No 34005100</td>
 </tr>
 <tr>
 <td>holo-Transferrin human</td>
 <td>Sigma</td>
 <td>Cat No T0665</td>
 </tr>
 <tr>
 <td>Insulin human</td>
 <td>Sigma</td>
 <td>Cat No I2643</td>
 </tr>
 <tr>
 <td>Gencin&reg;</td>
 <td>Delta Select</td>
 <td>Art No 7395800</td>
 </tr>
 <tr>
 <td>Recombinant Human Interleukin-4</td>
 <td>Immunotools</td>
 <td>Cat No 11130045</td>
 </tr>
 <tr>
 <td>Cyclosporin A</td>
 <td>Novartis</td>
 <td>Cat No NDC 0078-0109-01</td>
 </tr>
 <tr>
 <td>FBS</td>
 <td>LONZA</td>
 <td>Cat No DE14-802C</td>
 </tr>
 <tr>
 <td>HEPES Buffer</td>
 <td>PAA</td>
 <td>Cat No S11-001</td>
 </tr>
 <tr>
 <td>G-418 Sulphate</td>
 <td>PAA</td>
 <td>Cat No P02-012</td>
 </tr>
 <tr>
 <td>Trypsin/EDTA (10x)</td>
 <td>Invitrogen Gibco&reg;</td>
 <td>REF 15400-054</td>
 </tr>
 </tbody>
</table>
C. Miscellaneous Supplies:
<table cellspacing="0" border="1">
 <tbody>
 <tr>
 <td>Reagent</td>
 <td>Company</td>
 <td>Order No</td>
 </tr>
 <tr></tr>
 <tr>
 <td>Sterile pipette tips</td>
 <td>Sarstedt</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>6-well plate</td>
 <td>NUNC</td>
 <td>Cat No 140675</td>
 </tr>
 <tr>
 <td>50mL conical tube</td>
 <td>BD Falcon&trade;</td>
 <td>Cat No 352070</td>
 </tr>
 <tr>
 <td>Tissue culture flask</td>
 <td>SARSTEDT</td>
 <td>Cat No 83.1813.002</td>
 </tr>
 </tbody>
</table>

generation of human cd40-activated b cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

Watch this Scientific Journal Video about Generation of Human CD40-activated B cells at JoVE.com

Generation of Human CD40-activated B cells

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

generation-of-human-cd40-activated-b-cells

Research

JoVE Journal

Biology

15.2K 조회수.  University Hospital of Cologne, Department I of Internal Medicine. 이 비디오에서는 소개 예 생체내 생성 및 확장 (CD40 - B).

비디오: Generation of Human CD40-activated B cells

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Robust Generation of Hepatocyte-like Cells from Human Embryonic Stem Cell Populations

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical studies are substantial, therefore it is essential that more predictive toxicology screens are developed and deployed early on in drug development (Greenhough et al 2010). Human hepatocytes represent the current gold standard model for evaluating drug toxicity, but are a limited resource that exhibit variable function. Therefore, the use of immortalised cell lines and animal tissue models are routinely employed due to their abundance. While both sources are informative, they are limited by poor function, species variability and/or instability in culture (Dalgetty et al 2009). Pluripotent stem cells (PSCs) are an attractive alternative source of human hepatocyte like cells (HLCs) (Medine et al 2010). PSCs are capable of self renewal and differentiation to all somatic cell types found in the adult and thereby represent a potentially inexhaustible source of differentiated cells. We have developed a procedure that is simple, highly efficient, amenable to automation and yields functional human HLCs (Hay et al 2008 ; Fletcher et al 2008 ; Hannoun et al 2010 ; Payne et al 2011 and Hay et al 2011). We believe our technology will lead to the scalable production of HLCs for drug discovery, disease modeling, the construction of extra-corporeal devices and possibly cell based transplantation therapies.

This article will focus on the generation of human hepatic endoderm from human embryonic stem cell populations.

Despite progress in modelling human drug toxicity, many compounds fail during clinical trials due to unpredicted side effects. The cost of clinical ...

Phenotypic and Functional Characterization of Endothelial Colony Forming Cells Derived from Human Umbilical Cord Blood

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of circulating endothelial progenitor cells (EPCs) were first identified in adult human peripheral blood by Asahara et al. in 1997 2 bringing an infusion of new hypotheses and strategies for vascular regeneration and repair. EPCs are rare but normal components of circulating blood that home to sites of blood vessel formation or vascular remodeling, and facilitate either postnatal vasculogenesis, angiogenesis, or arteriogenesis largely via paracrine stimulation of existing vessel wall derived cells3. No specific marker to identify an EPC has been identified, and at present the state of the field is to understand that numerous cell types including proangiogenic hematopoietic stem and progenitor cells, circulating angiogenic cells, Tie2+ monocytes, myeloid progenitor cells, tumor associated macrophages, and M2 activated macrophages participate in stimulating the angiogenic process in a variety of preclinical animal model systems and in human subjects in numerous disease states4, 5. Endothelial colony forming cells (ECFCs) are rare circulating viable endothelial cells characterized by robust clonal proliferative potential, secondary and tertiary colony forming ability upon replating, and ability to form intrinsic in vivo vessels upon transplantation into immunodeficient mice6-8. While ECFCs have been successfully isolated from the peripheral blood of healthy adult subjects, umbilical cord blood (CB) of healthy newborn infants, and vessel wall of numerous human arterial and venous vessels 6-9, CB possesses the highest frequency of ECFCs7 that display the most robust clonal proliferative potential and form durable and functional blood vessels in vivo8, 10-13. While the derivation of ECFC from adult peripheral blood has been presented14, 15, here we describe the methodologies for the derivation, cloning, expansion, and in vitro as well as in vivo characterization of ECFCs from the human umbilical CB.

Endothelial colony forming cells (ECFCs) are circulating endothelial cells with robust clonal proliferative potential that display intrinsic in vivo vessel forming ability. Phenotypic and functional characterization of outgrowth endothelial cells derived from CB are important to identify and isolate bona fide ECFCs for potential clinical application in repairing damaged tissues.

Longstanding views of new blood vessel formation via angiogenesis, vasculogenesis, and arteriogenesis have been recently reviewed1. The presence of ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

Generation of Human Cardiomyocytes: A Differentiation Protocol from Feeder-free Human Induced Pluripotent Stem Cells

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is essential first to generate functional human cardiomyocytes (CMs). The use of these cells in drug discovery and toxicology studies would also be highly beneficial, allowing new pharmacological molecules for the treatment of cardiac disorders to be validated pre-clinically on cells of human origin. Of the possible sources of CMs, induced pluripotent stem (iPS) cells are among the most promising, as they can be derived directly from readily accessible patient tissue and possess an intrinsic capacity to give rise to all cell types of the body 1. Several methods have been proposed for differentiating iPS cells into CMs, ranging from the classical embryoid bodies (EBs) aggregation approach to chemically defined protocols 2,3. In this article we propose an EBs-based protocol and show how this method can be employed to efficiently generate functional CM-like cells from feeder-free iPS cells.

Pluripotent stem cells, either embryonic or induced pluripotent stem (iPS) cells, constitute a valuable source of human differentiated cells, including cardiomyocytes. Here, we will focus on cardiac induction of iPS cells, showing how to use them to obtain functional human cardiomyocytes through an embryoid bodies-based protocol.

In order to investigate the events driving heart development and to determine the molecular mechanisms leading to myocardial diseases in humans, it is ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...

Generation and Culture of Blood Outgrowth Endothelial Cells from Human Peripheral Blood

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular mechanisms underlying endothelial dysfunction in these individuals. However, the recent identification of blood outgrowth endothelial cells (BOECs), generated from circulating endothelial progenitors in adult peripheral blood, may circumvent this limitation by offering an endothelial-like, primary cell surrogate for patient-derived endothelial cells. Beyond their value to understanding endothelial biology and disease modeling, BOECs have potential uses in endothelial cell transplantation therapies. They are also a suitable cellular substrate for the generation of induced pluripotent stem cells (iPSCs) via nuclear reprogramming, offering a number of advantages over other cell types. We describe a method for the reliable generation, culture and characterization of BOECs from adult peripheral blood for use in these and other applications. This approach (i) allows for the generation of patient-specific endothelial cells from a relatively small volume of adult peripheral blood and (ii) produces cells that are highly similar to primary endothelial cells in morphology, cell signaling and gene expression.

This protocol allows for the reliable generation and characterization of blood outgrowth endothelial cells (BOECs) from a small volume of adult peripheral blood. BOECs can be used as a surrogate for endothelial cells from patients with vascular disorders and as a substrate for the generation of induced pluripotent stem cells.

Historically, the limited availability of primary endothelial cells from patients with vascular disorders has hindered the study of the molecular ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...