Regenerative medicine advance: 140 Cell Types From Human Embryonic Stem Cells

Research published this week in Regenerative Medicine, reports on a new technology that yields over 140 previously uncharacterized cell types, many on an industrial scale. This advance holds great promise for future research and may one day lead to many new cell-based therapies in the emerging field of regenerative medicine.
Previously there have been occasional reports that individual cell types have been generated from hES (human embryonic stem cells) cells, such cells have often been generated in small quantities, not useful on an industrial scale.

In a paper titled The ACTCellerate Initiative: large-scale combinatorial cloning of novel human embryonic stem cell derivatives”, a team led by Dr Michael D. West, now CEO, BioTime, Inc and Adjunct Professor, University of California, Berkeley, along with collaborators at Advanced Cell Technology, the Burnham Institute, Ontario Cancer Institute, and the University of California, San Francisco demonstrated that primitive precursors of the many body cell types have an unpredicted ability to be propagated from a single cell, leading to the clonal expansion of these embryonic progenitor cell types. A careful genome-wide analysis of gene expression showed evidence that the “Zip code” that the developing body uses to place cells in their proper location in the body is preserved in these cells, giving researchers a means to make cell types from a single location in the body.

Another important finding in this publication is that these highly purified cell types show that primitive embryonic cell types show the expression of genes generally associated with malignant cancer. However, when used in this highly purified form, no malignant tumors could be observed when the cells were injected into mice.

Dr Chris Mason (UCL), Associate Editor of Regenerative Medicine said, “This is an enormously exciting development for the regen sector. The research reported by Dr West and his team represents a quantum leap forward in embryomics, the mapping and characterization of the cells of early human development. Without any doubt, the ACTCellerate technology will greatly hasten the translation of human embryonic stem cell-based therapies into safe and effective products for routine clinical practice “.

“The demonstration that combinatorial cloning can lead to numerous and diverse purified cell types opens the door strategies to map the human embryome. This roadmap is critical to the clinical application of the emerging field of regenerative medicine”, said Dr. West.


The unique challenges of pluripotency

• Human embryonic stem (hES) cells show the capacity to differentiate into all of the hundreds of somatic cell lineages in the developing human.

• Human embryonic progenitor (hEP) cells are primitive precursors of terminally differentiated cells that are capable of propagation in vitro and display makers generally associated with the embryonic stages of development.

• Challenges to the field include the mapping of the ‘embryome’ for example to identify the unique molecular markers that allow the identification and isolation of hEP cells.

Multiplex generation & characterization of hEP cell clones

• The ACTCellerate protocol utilizes a two-step differentiation and propagation protocol to isolate clonal populations of scalable hEP cell lines. Of 1090 clones isolated in this report, 280 lines (25.7%) expanded to at least four roller bottles.

Clonal hEP cells do not display hES markers but instead show markers of diverse primitive embryonic progenitors

• Cell lines derived with the ACTCellerate protocol do not express markers of hES cells such as hTERT or OCT4.

• A total of 71% of the hEP cell lines show positive expression for MEOX1, MEOX2, or FOXF1, which in the mouse are reported to be expressed only in early stages of embryonic development.

• Non-negative matrix factorization suggested that the complexity of distinct cell types isolated was at least 140.

Immunocytochemical confirmation of hEP microarray gene-expression analysis

• Selected markers in putative ectodermal, mesodermal, endodermal and neural crest gene cell lines were confirmed by immunocytochemistry.

Clonal hEP lines express diverse cell surface antigens

• Flow cytometry confirmed that gene expression often predicts CD antigen expression on the cell surface and provides a means of manipulating hEP cell types.

hEP clones express unique secreted factors

• hEP cell clones expressed diverse secreted growth factors. Select factors confirmed by ELISA included AREG, FGF7, IGFBP5, TGFβ-1 and PDGF-BB.

hEP cells lack tumorigenicity

• hEP cell lines expressed a wide array of oncofetal genes including: SILV, PLAG1, AMIGO2, HCLS1, SPINK1, PRAME, INSM1, ENC1 and CEACAM1, as well as others.

• Clonal hEP cell lines did not generate malignant tumors in 4-6 months at doses of 10 million cells per mouse when injected subcutaneously and intramuscularly.

hEP cells include clones with a robust & mortal proliferative capacity

• hES cells express high levels of telomerase activity by telomeric repeat amplification protocol (TRAP) assay, and an immortal phenotype when maintained in the undifferentiated state.

• hEP cells were telomerase negative by TRAP assay, but often display a long proliferative lifespan in vitro useful in scaling the cells.