Picture Blog: A Short Path from Human mRNA-iPSCs to Neurons in Record Speed

Traditional differentiation protocols use embryoid body (EB) formation as the first step of lineage restriction to mimic early human embryogenesis, which is then followed by manual selection of neuroepithelial precursors. This procedure is tedious and often inconsistent. We have developed a novel neural differentiation scheme that directs human iPSCs (created with the Allele 6F mRNA reprogramming kit) that progressed, as attached culture, to neural precursor cells (NPCs) in just 4-6 days, half the time it typically takes by other methods. From NPCs it takes about another 5-6 days for neural rosettes to form (see figures below); upon passage, cells in neural rosettes differentiate into neurons in 24 hours.

The neural progenitors at the rosettes stage can be stocked and expanded, before differentiated into different types of neurons. We are working on specifically and efficiently different these neural progenitor cells into dopaminergic, glutamatergic, GABAergic, and other types of sub-types of neurons with Allele’s technologies (Questions? email the Allele Stem Cell Group at iPSatAllelebiotech.com).

Tags: differentiation, dopaminergic, EB, GABAergic, glutamatergic, iPSC, mRNA, neural rosettes, neuron, regenerate medicine

Monitoring the Undifferentiated Stage of Stem Cells—the Pluripotency Markers

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 are very difficult tasks 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.

To identify pluripotency of stem cells, expression of stem cell-specific marker genes (i.e. Oct-3/4, Sox2, Nanog, Rex-1) is monitored by RT-PCR. Alkaline phosphatase activity and methylation profiles of promoters of pluripotency-relevant genes are often analyzed as well. Compared to murine cells, it is noticeably more difficult to obtain human iPSCs, of which stem cell-like colonies sometimes turn out not to be pluripotent cells. We highly recommend testing iPSCs, especially human iPSCs, with antibodies against stage-specific embryonic antigens such as SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

However, all of these methods require cell destruction or fixation for analysis, therefore, are inconvenient and costly. Furthermore, many studies using ES or iPS cells involve differentiation of stem cells into different lineages, a method for observing live cells to know their undifferentiation/differentiation stages would be very helpful. There have been a number of publications using murine Oct-4, Nanog, and Rex-1 promoter 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 have been reported.

New product of the week 01-25-10 to 01-31-10:

All-In-One-Vector: Human OSKM Lentiviral Paticles, with Oct-4, Sox-2, Klf, and c-Myc all expressed from a single virus, ready-to-use.

• Promotion of the week:

human iPS cell detection primer set, the same as the landmark Yamanaka paper [4] on creating human iPS for the first time.

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
4. Takahashi et al, Induction of Pluripotent Stem Cell from Adult Human Fibroblasts by Defined Factors (2007). Cell 131, 861-872

Tags: differentiation, fluorescent protein, iPS, iPS lentivirus, iPSC, iPSCs, lentivirus, pluripotency reporter, promoter, promoter-FP, stem cells, undifferentiation stage