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Stem Cells

 

Non-Integrating iPSC Generation

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Allele Biotechnology's NextGen mRNA platform allows for easy, hightly efficient, feeder free and xeno free iPSC generation in less time than every before.  We are offering three unique ways for you to harness this breakthrough in iPSC reprogramming:

  • 6F mRNA reprogramming premix:  A validated ready-to-use mRNA mixture with 6 factors including an engineered Oct4 for accelerated iPSC induction
  • Custom iPSC Generation Service:  A fully customized all encompassing service for your specific cell type, including target cell culture, reprogramming and validation
  • IVT Templates: Validated ready-to-use IVT templates for generating your own mRNA, reprogramming and directed differentiation factors are available

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References:

    1. Warren, L. et al. Feeder-Free Derivation of Human Induced Pluripotent Stem Cells with Messenger RNA. Scientific Reports, srep00657 (2012)

 

iPSCs Generation

Induced Pluripotent Stem Cells (iPSC): Stem Cell Generation

The direct reprogramming of somatic cells to pluripotency was first accomplished in 2006. Adult mouse fibroblasts were converted to iPS Cells (induced pluripotent stem cells) through ecotropic expression of a select group of transcription factors. In 2007, direct reprogramming was achieved in human cells. The generation of iPS from differentiated adult cells has vast therapeutic implications, particularly in the context of pharmaceutical screening and cellular replacement therapies. Extensive research has been conducted in search of efficient ways for stem cell generation.

In this system, 4 product lines are available:

      1. Basic Lentiviral Particles: lentiviruses carrying each of the 6 reported reprogramming factors, hOct3/4, hSox2, hKlf4, hc-Myc, hNanog and hLin28, or enhancing factors like p53 shRNA and hTert are provided individually or in combination sets.
      2. Lentiviral Particles co-expressed with Reporter: fluorescent proteins are co-expressed from the iPS factor carrying lentivirus to trace their expression during the re-programming procedure.
      3. 2-in-1 OS and 4-in-1 OSKM with loxP Sites. Polycistronic expression of hOct3/4, hSox2, hKlf4 and hc-Myc, in one lentiviral vector, referred to as 4-In-1, reported reprogramming efficiently. More recently, polycistronic expression of only two of the essential factors, hOct3/4 and hSox2, referred to as 2-In-1, is gaining popularity in iPSC generation. Meanwhile, it seems beneficial to silence the exogenous iPS factors after the initiation of reprogramming. Allele Biotech combines all these advancements in iPSCs field and provides both the 4-In-1 and 2-In-1 with loxP sites by which the iPS factors may be removed with a Cre recombinase.
      4. Basic Retroviral Particles: MMLV-based retroviruses carrying each of the 6 reported reprogramming factors, hOct3/4, hSox2, hKlf4, hc-Myc, hNanog and hLin28 are provided individually or in combination sets. Retroviral systems are preferred by some iPSC researchers because retrovirus is silenced by the host more efficiently than lentivirus after the trangenes exert their effects.

To learn tips about growing iPS cells or dealing with iPS products, email us at ips@allelebiotech.com or check out the latest in our iPS blog.

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References:

      1. Takahashi, K., and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult Fibroblast cultures by defi ned factors. Cell 126, 663–676.
      2. Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., and Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872.
      3. Maherali, N., and Hochedlinger, K. (2008). Guidelines and techniques for the generation of Induced pluripotent stem cells. Cell Stem Cell 3

 

Basic lentiviral particles

Lentiviral vectors were used in the original iPSC experiments and remain to be the most popular method. Its advantages over other gene-delivery methods include high-efficiency infection of dividing and non-dividing cells, long- term stable expression of transgenes, and low immunogenicity. Allele Biotech provides custom retrovirus or lentivirus packaging services using proprietary technologies that are unique and highly efficient, yielding 10^8 to 10^9 TU/ml, without any concentrating steps. These technologies and optimal operation procedures enable us to package viruses at < 1% of the market price in certain categories on per million particle basis. We offer Pre-packaged iPSCs Generation Lentiviral Particles, which are validated, ready-to-use, high quality reagents for any laboratory to create iPS cells. A straight-forward and optimized protocol is also provided for immediate use of these products.

For additional information, email us at ips@allelebiotech.com or check out the latest in our iPS blog.

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2-in-1 OS and 4-in-1 OSKM Lentiviral Particles

Putting 4 iPS factors on one lentiviral vector, separated by 2A peptides, has appeared to be more efficient in generating iPS cells than having all 4 factors on individual viruses, at least in a number of cases. Stem cell-like colonies start to appear in about 2 weeks using Allele Biotech’s 4-in-1 lentivirus. In addition to the concerted effects from Oct3/4, Sox2, c-Myc, Klf4, it is also believed that the coordinated silencing of these factors after reprogramming helps to form iPS colonies. The 4-in-1 lentivirus from Allele Biotech contains loxP sites that can be used to remove the 4 cDNAs if so desired.

For additional information, email us at ips@allelebiotech.com or check out the latest in our iPS blog.

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iPSCs Validation

Nanog, Oct4, and Rex1 promoters have been reported to display ES/iPS cell specific expression under undifferentiated states. Allele Biotech provides pre-packaged lentiviral fluorescent protein (FP) reporters driven by all three promoters to confirm the state of the reprogrammed iPSCs. A GFP or RFP gene was cloned behind these ES-specific promoters to offer convenient fluorescence tracking of undifferentiated cells.

We also provide the iPS Cell Identification CR Primer Kits. Instead of ordering individual oligos, complete sets of primers, as precisely defined and tested in Takahashi et al. 2006, are conveniently and sufficiently provided for 50 reactons for each gene.

For iPSCs identification antibodies, chech the Antibody group or search in products.

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References

      1. Da Yong WU, Zhen YAO (2005). Isolation and characterization of the murine Nanog gene promoter. Cell Research, 15 (5): 317–324.
      2. Rachel Eiges, Maya Schuldiner..et.al (2001). Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cell. Current Biology 11: 514–518.
      3. Guangjin Pan, Jun Li, Yali Zhou, Hui Zheng, and Duanqing pei (2006). A negative feedback loop of transcription factors that control stem cell pluripotency and self renewal. ASEB Journal 20: E1094~E1102

Note: The specificity of these promoters is based on scientific publications, which are subject to debate.

 

Pluripotency Reporters

Human embryonic stem (ES) cells or induced pluripotent stem (iPS) cells promise to serve as an unlimited source for transplantation or tissue-specific differentiation. However, obtaining and maintaining stem cells can be difficult for multiple reasons. For instance, most stem cell lines tend to spontaneously differentiate in culture, and even if the cells form stem cell-like colonies, they may be of a heterogeneous population. There have been a number of publications using murine Oct-4, Nanog, and Rex-1 promoters driven fluorescent proteins as markers for pluripotency tests [1-3]. Allele Biotech provides, under its iPS product line, packaged and validated lentiviral particles that would insert these 3 promoter-FP reporters into the stem cells. Although currently these promoters are of mouse sequences, their use in human stem cells has been reported.

To learn more about pluripotency reporters, email us at ips@allelebiotech.com or check out the iPS Pluripotency Reporter blog entry.

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Feeder Cells: Human Fibroblast and MEF Feeder Cells

Allele offers pre-irradiated, ready-to-use, system specific, bFGF-Producing Feeder Cells for iPSC propagation. Using Allele’s bFGF-Producing Feeder Cells avoids the usual problems associated with MEF cell lines. They are maintained at low passages, come pre-irradiated and exogenously express bFGF, so there is no need to supplement your medium with additional growth factors. Additionally, Allele is introducing human fibroblasts to the market for iPSC work. MEF is good for mouse iPSC reprogramming but human fibroblast feeders are preferred when creating human iPSCs due to their secreted factors. Propagate human iPSC with greater efficiency while eliminating non-human cells for therapeutic use of human iPSCs.

About Feeder Cells

Pluripotent stem cells can be used to generate virtually all types of differentiated cells for research, drug testing, or therapeutic development. Maintaining embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC) karyotypically normal and in a pluripotent, self-renewing state for a reasonable and defined period of time currently requires culturing ESC or iPSC in contact with established feeder cells such as mouse embryonic fibroblasts (MEFs) or pooled extracellular matrix (ECM) products such as "Matrigel."

About MEF and LIF

Mouse embryonic fibroblasts (MEF) expressing murine bFGF and LIF, are irradiated for direct plating as feeder cells to support iPSC generation using mouse cells. Leukemia inhibitory factor (LIF) is a 20 kDa protein that is known for its ability to inhibit the differentiation of embryonic stem cells in mice and contribute to stem cell selfrenewal. Human and mouse LIF proteins share 79% sequence homology and exhibit cross-species activity. However, LIF inhibition of stem cell differentiation appears to be mouse-specific.

      • Already irradiated and tested, ready-to use
      • MEF LIF for maintaining mouse iPSCs
      • Expressing bFGF, no need to supply recombinant bFGF as a medium supplement

For additional information, email us at ips@allelebiotech.com or check out the latest in our iPS blog.

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