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Being able to genetically manipulate hESCs and their derivatives opens new avenues in the study of human embryogenesis and the development of future cell therapies. Human ESCs provide a unique opportunity to study molecular mechanisms that regulate specification of the hematopoietic lineage in the human. Exploitation of this model using transgenic strategies depends on the ability to effectively target cells of the hematopoietic lineage and establish stable transgene expression. Mouse and hES derivatives are, so far, ineffective at repopulating hematopoiesis in lethally irradiated adults (15,16). Previous data suggest that mouse primitive embryonic HSC can be induced to become definitive lymphoid-myeloid HSC if exposed to the proper microenvironment (24). Recently, it has been shown that mouse primitive HSC can become definitive HSC by retroviral overexpression of either HoxB4 or Stat5 (23,33). The generation of NOD/SCID repopulating cells (SRC) from hESC might be promoted by some of these genes as well as other potential factors capable of regulating primitive blood cells and inducing human stem cell expansion such as HoxB4

Fig. 3. Assessment of transduction efficiency into hematopoietic progenitors by using polymerase chain reaction (PCR) for proviral integration. Representative agarose gel stained with ethidium bromide containing the amplified PCR products from a GFP PCR done with individually plucked colonies. Positive DNA control can be extracted from PG13-GFP and an appropriate negative control is DNA extracted from PG13-Mock and mock-infected colonies. To ensure the availability of amplifiable template, DNA extracted from colonies should be subjected to a human-specific gene such as CART-1.

Fig. 3. Assessment of transduction efficiency into hematopoietic progenitors by using polymerase chain reaction (PCR) for proviral integration. Representative agarose gel stained with ethidium bromide containing the amplified PCR products from a GFP PCR done with individually plucked colonies. Positive DNA control can be extracted from PG13-GFP and an appropriate negative control is DNA extracted from PG13-Mock and mock-infected colonies. To ensure the availability of amplifiable template, DNA extracted from colonies should be subjected to a human-specific gene such as CART-1.

(23), Bmi-1 (37,38) and different members of Notch (39,40), Wnt (41), or Hedgehog pathways (42). The successful delivery of a reporter gene (GFP) into CD45negPFV hemogenic precursors by means of a Gibbon ape leukemia virus (GALV)-pseudotyped retroviral gene transfer strategy paves the way for further gain-of-function studies conducted to address the potential role of different candidates genes in the generation of SRC from hESC and will provide a better understanding of the molecular mechanisms underlying cell fate.

4. Notes

1. PG13 is a retrovirus packaging cell line derived from TK- NIH/3T3 cells based on the GALV. Introduction of retroviral vectors by infection or transfection results in the production of retrovirus virions capable of infecting cells expressing GALV receptor 1 (also known Pit-1). CD45negPFV precursors express high levels of Pit-1 by RT-PCR (32).

2. The functional titer of our GFP-expressing PG13 packaging cell line is 5 x 105 infectious virus particles per milliliter. Although many parameters may influence the titer, between 2 x 105 and 1 x 106 infectious virus particles per milliliter is usually considered a suitable titer when working with PG13 cell line.

3. Nontransduced PG13 retroviral packaging cell line should be used as mock in each experiment.

4. Purity average should be between 90 and 100%.

5. Although viruses produced by GALV-pseudotyped PG13 cell lines will no longer be able to replicate after they infect any target cell, they still are capable of infecting human cells. Caution should always be exercised in the production and handling of any recombinant retrovirus. For this reason, we highly recommend that you treat retroviral stocks generated by following Biosafety Level 2 guidelines. We also recommend consulting the health and safety guidelines and officers at your institution before starting your retroviral experiments.

6. The protocol detailed is optimized for 96-well plates. However, if you have enough cells you may scale up the procedure by using 48- to 6-well plates as long as you proportionally increase all the volumes (e.g., 1X serum-free complete media, virus-containing supernatants) as needed and maintain the concentration of all different cytokines and reagents.

7. 10-d prestimulation liquid culture is optimal to get the highest transduction efficiency into CD45negPFV-derived hematopoietic progenitors with CFU potential. Shorter prestimulation periods impair the transduction of hematopoietic progenitors with CFU capabilities although it slightly enhances the transduction efficiency of overall hematopoietic cells (CD45+).

8. It contains DMSO from the freezing procedure, which can negatively affect cell growth.

9. Virus production from freshly thawed PG13 cells is poor, so it is strongly recommended to have cells growing well approx 1 wk before collection of virus-containing supernatant.

10. Fibronectin facilitates colocalization of virus particles and target cells increasing the likelihood of infection of target cells.

11. The volume of viral supernatant used to preload the plate (2 x 100 |L) and to resuspend the cells (200 |L) is adjusted in order to have a multiplicity of infection (MOI) between 3 and 5: if virus titer equals 5 x 105/mL and each well contains 200 | L of viral supernatant it means that each well would have five times the input number of cells originally seeded (2 x 104). Although we have not tried to further increase the MOI, it is likely that higher MOI provides higher trans-duction levels.

12. It should be very easy to harvest the cells with no need of enzymatic digestion because they grow loosely attached to the fibronectin. If by any chance they do not lift up easily, just add 25 ||L of 0.25% Trysin-1 mM EDTA and incubate for 1 min before blocking trypsin activity with 100 |L of IMDM + 10% FBS and harvest them.

13. We have experienced that isolated CD45negPFV precursors do not like to be repeatedly collected and transferred to a new fibronectin-coated, viral-preloaded well; resulting in a notable cell death. However, using the present retroviral gene transfer protocol we were able to get acceptable levels of transduction by precoating with fibronectin and preloading with viral supernatant just on the first day of retroviral exposure.

14. If no more experiments are scheduled for the next 7 d, it is strongly advised you discard the packaging cell line. Each laboratory should have enough frozen stocks of the packaging cell line for future retroviral gene transfer experiments. Prolonged culture of the PG13 cell line results in a drop of the titer and increases the likelihood for undesirable recombination events leading to the generation of helper-free infective virus (34-36).

15. Data from our laboratory (32) indicate that the addition of FBS to cultures after retroviral exposure supported transgene expression resulting in hematopoietic progenitors derived from CD45negPFV hemogenic precursors.

16. At the end of the in vitro culture of CD45negPFV hemogenic precursors, we generally observe a twofold cell expansion as compared with the number of cells originally seeded (32).

17. After this incubation you should not see any kind of cell pellet, debris, or white material floating in the tube.

18. All steps involving phenol or chloroform must be performed in a fume hood; and a lab coat, gloves, and safety glasses must be worn at all the times. Besides, an appropriate waste container for phenol and chloroform disposal is required.


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Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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