Human induced pluripotent stem (iPS) cells have the potential to greatly impact many areas of research and medicine and can be generated by reprogramming somatic cells through transient overexpression of key reprogramming factors.
While dermal fibroblasts were the first human cell type to be reprogrammed into iPS cells, blood cells are also increasingly being utilized as a starting cell type due to the limited invasiveness of sample collection, and the availability of banked blood samples representing a variety of disease, age, gender and geographical subtypes.
The choice of cell type to use for reprogramming is based on:
- The accessibility of tissue samples
- The genetic make-up of the target cells
- The reprogramming efficiency
Challenges of Reprogramming Blood Cells
One of the challenges of reprogramming blood cells is the low frequency of certain cell types in peripheral blood. Peripheral blood cell types have varying reprogramming efficiencies, where efficiency is often inversely correlated with frequency in blood. For example, CD34+ hematopoietic stem and progenitors cells have relatively high reprogramming efficiencies, but are rare in circulating blood. In a previous blog post: Reprogramming Blood Cells: How to Decrease Variability in Your Workflow, we discussed some of the solutions that STEMCELL Technologies has developed to isolate and expand rare cell types, such as erythroid and CD34+ progenitor cells, from peripheral blood in order to obtain sufficient numbers for reprogramming.
Another common observed phenomenon is the emergence of partially reprogrammed colonies, usually associated with the continued expression of reprogramming factors. These cells are phenotypically diverse and often fail tests of pluripotency. While overall efficiency is lower, iPS cells that emerged under feeder-free conditions were fully reprogrammed, indicating the importance of culture conditions in the reprogramming process.
Join Wing Chang, Scientist at STEMCELL Technologies in this Ask the Expert Session, as we discuss the technical challenges of reprogramming blood cells, considerations when choosing the somatic cell type to be reprogrammed, implications of starting cell type on reprogramming efficiency and downstream differentiation, and other reprogramming questions.
We have been generating iPSCs using episomal vectors can you tell me how your product would differ from this method and what are its advantages/disadvantages?
0 0 0 Total: 0 Our reprogramming media products (TeSR™-E7™, ReproTeSR™) are compatible with multiple vector systems such as episomal vectors. Our media products are optimized for feeder-free reprogramming and consist of defined components and xeno-free.
0 0 0 Total: 0 We currently recommend Corning® Matrigel® for reprogramming blood cells.
0 0 0 Total: 0 Chan et al (1) demonstrated that reprogramming efficiency under feeder-free conditions was lower, but that all of the iPS cells generated were fully (not partially) reprogrammed. Therefore, it seems that culture conditions have an important role in the reprogramming process. That is why we recommend STEMCELL Technologies’ feeder-free, defined, and … Continued
0 0 0 Total: 0 CD34+ progenitors were one of first sources of blood cell types to be reprogrammed, though more recently, erythroid progenitors have been gaining more widespread use.
0 0 0 Total: 0 Blood cells are continuously produced from stem cells in the bone marrow. The use of blood cells as a source for generating iPS cells is gaining more widespread interest due to the ease in accessibility. Some popular blood cell types used are CD34+ progenitors, erythroid progenitors, T- and B-cells. However, … Continued
In reprogramming cord blood cells, we have found that our cells are detaching, thus negatively affecting our reprogramming efficiency. Any suggestions?
0 0 0 Total: 0 It is difficult to say what may be causing detachment of cells and whether the cells that are detaching are blood cells that are not being reprogrammed and are dying over long-term culture. If the cells that are detaching are pre-iPS cell-like colonies, I would re-examine the vector system and … Continued
0 0 0 Total: 0 We typically perform directed differentiation of iPSCs (generated from blood cells) using monolayer cultures. For examples, please refer to our STEMdiff Neural Induction Medium or STEMdiff Definitive Endoderm kits for our monolayer protocols. However, should you decide to differentiate using embryoid bodies (EBs), we would recommend using Aggrewell™ plates for … Continued
Do you have a protocol for reprogramming with episomal vectors? Is this preferred to sendai virus reprogramming?
0 0 0 Total: 0 Episomal vectors and Sendai virus are perhaps the two most common vector systems currently being used. Both systems work well and have been adopted by numerous labs (see Schlaeger et al., Nat Biotechnol, 2015) but preference will typically come down ease-of-use, cost, and downstream analysis for vector clearance or integration. … Continued
Can you speak to the advantages and challenges of using blood cells to generate iPSCs versus other cell types?
0 0 0 Total: 0 A couple of advantages of using blood cells as a source for generating iPSCs include the ease of obtaining blood samples from donors, and the higher reprogramming efficiency of blood cells compared to other cell types. One of the challenges of using blood cells is determining which blood cell type … Continued
What methods are available for reprogramming blood cells into iPS cells that don’t include the introduction of foreign genetic material? Are there limitations to these methods?
0 0 0 Total: 0 There currently are no robust methods available to generate iPS cells without the introduction of foreign genetic material such as DNA or RNA. That being said, RNA-based vector systems cannot integrate, therefore no exogenous genetic material will be introduced into the genome.
0 0 0 Total: 0 Pluripotency can only be confirmed by tri-lineage differentiation either through in vivo formation of a teratoma containing all three germ layers or by in vitro differentiation to cells of each germ layer. Our preferred method would be in vitro directed differentiation using: STEMdiff™ Definitive Endoderm kit for differentiation, STEMdiff™ NIM … Continued