Cord blood hematopoietic stem cells expansion – what is the best starting population?

by Alexey Bersenev on August 7, 2012 · 0 comments

in cord blood, hematopoietic

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Expansion of hematopoietic stem/ progenitor cells (HSPC) from cord blood is a promising direction to increase donor cell engraftment and improve outcome. Variety of strategies for HSPC ex vivo expansion have been explored in clinic. But most of protocols are not robust and easily reproducible. One of the controversies is defining the best starting population for ex vivo expansion. It was proposed that such cord blood cell populations could be used in clinic as: total mononuclear cells (MNC), CD3 and CD14 depleted MNC, Lineage-negative (Lin-), sorted or enrich CD34+ cells and CD133+ cells. The recent study, published in Transfusion, can shed more light on this controversy.

The authors compared ex vivo expansion kinetics of 5 different starting cell populations, derived from cord blood:

  1. Total MNC (~ 0.9% CD34+)
  2. Enriched by negative selection CD34+/CD38+ (~ 59% CD34+)
  3. Enriched by negative selection CD34+/CD38- (~ 74% CD34+)
  4.  Sorted pure CD34+/CD38+ (>98% CD34+)
  5. Sorted pure CD34+/CD38- (>98% CD34+)

The results of this study are very important and should be considered by any clinical group. Brief summary:

1. MNC was the worst starting population, because expansion of HSPC was not observed. Colony-forming ability and number of CD34+ cells, derived from cultured MNC, declined rapidly. This observation confirmed the notion, that accessory cells can inhibit HSPC expansion.

2. Purified CD34+/CD38- was the best starting population in terms of expansion potential and colony-forming ability:

… based solely on their intrinsic proliferation and expansion capacities, it is clear that Population V (consisting of a pure population of CD34+ CD38− cells, reaching 12,800-fold increase in total cells, 1280-fold increase in CD34+ cells, 490-fold increase in CFCs, and 12-fold increase in LTC-ICs) would be the most suitable population for clinically oriented ex vivo expansion.

3. All populations (2-5), enriched for CD34+ cells, performed very well in culture for 4 weeks and, at some time points, didn’t demonstrate significant difference in colony-forming assay or CD34+ cell number. It suggests, that any population, enriched or purified for CD34+ (negative/ positive magnetic selection or fluorescent sorting), could be good input for clinical-grade expansion. These data in agreement with other reports, which were not able to detect significant difference between CD34+ total and CD34+/CD38- starting populations.

4. The authors suggest to culture cord blood-derived HSPC for 10-14 days. It could be the best time for cell harvest, because at time point of 2 weeks CD34+ enriched populations are reaching sub-maximal potential and proliferation kinetics. Also, too much manipulations, associated with long-term culture, is not recommended.

The advantage of this study is using stroma-free and serum-free protocol. It seem to me that the protocol could be easily reproducible. The disadvantage is the absence of transplantation assays, which allow to tack HSPC functionality in vivo.

In summary, in this study we have shown that under our culture conditions, significant expansion of both HSCs and HPCs can be achieved in cultures of enriched or purified CD34+ cell populations. Our study suggests that the cell fraction containing more than 98% CD34+ CD38− cells would be the ideal one to achieve maximum expansion in large-scale protocols; however, based on our data, it seems that other cell populations—enriched for CD34+ cells—would also be appropriate as input cell fractions for clinically oriented ex vivo cell expansion.

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