Production of stem cells with embryonic characteristics from human umbilical cord blood

Abstract

When will embryonic stem cells reach the clinic? The answer is simple -- not soon! To produce large quantities of homogeneous tissue for transplantation, without feeder layers, and with the appropriate recipient's immunological phenotype, is a significant scientific hindrance, although adult stem (ADS) cells provide an alternative, more ethically acceptable, source. The annual global 100 million human birth rate proposes umbilical cord blood (UCB) as the largest untouched stem cell source, with advantages of naive immune status and relatively unshortened telomere length. Here, we report the world's first reproducible production of cells expressing embryonic stem cell markers, - cord-blood-derived embryonic-like stem cells (CBEs). UCB, after elective birth by Caesarean section, has been separated by sequential immunomagnetic removal of nucleate granulocytes, erythrocytes and haemopoietic myeloid/lymphoid progenitors. After 7 days of high density culture in microflasks, (10(5) cells/ml, IMDM, FCS 10%, thrombopoietin 10 ng/ml, flt3-ligand 50 ng/ml, c-kit ligand 20 ng/ml). CBE colonies formed adherent to the substrata; these were maintained for 6 weeks, then were subcultured and continued for a minimum 13 weeks. CBEs were positive for TRA-1-60, TRA-1-81, SSEA-4, SSEA-3 and Oct-4, but not SSEA-1, indicative of restriction in the human stem cell compartment. The CBEs were also microgravity--bioreactor cultured with hepatocyte growth medium (IMDM, FCS 10%, HGF 20 ng/ml, bFGF 10 ng/ml, EGF 10 ng/ml, c-kit ligand 10 ng/ml). After 4 weeks the cells were found to express characteristic hepatic markers, cytokeratin-18, alpha-foetoprotein and albumin. Thus, such CBEs are a viable human alternative from embryonic stem cells for stem cell research, without ethical constraint and with potential for clinical applications.

Figure 1

Cord blood‐derived embryonic‐like stem cell morphology. CBEs were identified as early as week 1 of liquid culture as tight clustered colonies which progressively increased in size and number.

Figure 2

Cord blood‐derived embryonic‐like stem cells’ proliferation profile. CBEs exhibited a four‐stage proliferation pattern: (i) a lag phase (baseline to week 1); (ii) exponential phase (week 2 to week 7); (iii) reduction in proliferation (week 7 to week 9); (iv) plateau/maintenance phase (week 9 to week 14). CBEs could be expanded 389‐fold from baseline at week 7.

Figure 3

Cord blood‐derived embryonic‐like stem cells’ production of second generation colonies. First‐generation CBEs were dissociated and subcultured yielding 168‐fold expansion from baseline for 6 weeks.

Figure 4

Cord blood‐derived embryonic‐like stem cells expression of embryonic stem cell‐specific markers. CBEs expressed embryonic sialoproteins SSEA‐3, SSEA‐4, extracellulamatix components Tra 1‐60, Tra 1‐81, and the pluripotency transcription factor Oct‐4 (scale bar 20 µm).

Figure 5

Cord blood‐derived embryonic‐like stem cells in 3‐dimensional hepatic tissue bio‐engineering. CBEs were dispersed and grown in a Synthecon microgravity simulating rotating cell culture system microbioreactor (top left quadrant). In this 3‐dimensional shear stress‐free environment cell growth was maintained until week 3. The observed decrease in single cell numbers was explained by the formation of 3‐dimensional hepatic aggregates (top right quadrant). Phenotypic analysis confirmed that CBEs had successfully differentiated into hepatic progenitors (bottom quadrant, scale bar 20 µm).