Human CD34-negative hematopoietic stem cells

by Alexey Bersenev on December 18, 2010 · 0 comments

in under discussion

CD34 is a commonly used and clinically significant marker for human hematopoietic progenitors and stem cells. Flow cytometric separation of distinct subsets within the CD34+ population is used to create a comprehensive map of human hematopoietic progenitor and stem cells. The possibility of separating different progenitor/ stem cell subsets could be used in clinic for differential mobilization, outcome prediction and prognosis, graft manipulation and engineering. Nevertheless, It was shown more than a decade ago that there are hematopoietic stem cells (HSC) in CD34-negative population in human. I was wondering what has changed since then and how can we apply our knowledge about CD34-negative HSC?

Confirmation of HSC existence in CD34-negative population
In 1998 Zanjani for the first time identified stem cell activity in CD34-negative fraction of Lineage-negative (Lin-) bone marrow cells, based on the human/sheep xenogeneic model. In the same time Mickie Bhatia from John Dick lab showed that CD34-negative cells (which are also Lin-) isolated from bone marrow and cord blood, are capable of long-term multilineage repopulation in SCID mice (so-called SCID-repopulating cells – SRC). One year earlier, Margaret Goodell, isolated so-called side population (SP) HSC, which were negative for CD34.

Studying stem cell activity
Bhatia studied two subsets of Lin-/CD34- HSC, based on expression of CD38. Both subsets repopulated SCID/NOD mice in long-term, but CD38+ much less. Magically, Sonoda group was not able to reproduce SCID/NOD mice repopulation by intavenous injection (IV) of Lin-/CD34- cells, derived from cord blood (CB). In their hands, CB-derived CD34- cells repopulated mice only via intra-bone transplantation.

Sonoda group tested self-renewal capability of cord blood-derived CD34-negative cells (via intra-bone route) and confirmed that it was comparable with CD34+ HSC.

Limiting dilution transplantation assay for CD34-negative HSC activity was performed by several groups. Recently, Sonoda’s group has developed very sensitive transplantation assay – intra-bone injection in NOG mice. Sensitivity of this assay and improved purification technique is based on the 18 Lineage antibody mixture, allowing the calculation of the frequency of CD34-negative HSC to be as high as 1 in 1000. Overall, frequency of HSC in CD34-negative is much inferior comparing with CD34+ counterpart. I summarized some experimental data below:

IV injection:
1 SRC in fresh 125 000 Lin-/CD34- (Bhatia, 1998)
1 SRC in cultured 38 000 Lin-/CD34- (Bhatia, 1998)
1 SCR in fresh 660 Lin-/CD34+/CD38- (Yahata et al, 2003)

Intra-bone injection:
1 SRC in 1010 Lin-/CD34+ (Wang, 2003)
1 SRC in 24100 Lin-/CD34- (13 antibodies used for Lineage detection, Wang, 2003)
1 SRC in 1000 Lin-/CD34- (18 antibodies used for Lineage detection, Ishii, 2010)
1 SCR in 40-44 Lin-/CD34+/CD38- (Ishii, 2010; Yahata et al, 2003)

Functional similarities between CD34+ and CD34- cells

1. CD34-negative HSC can be mobilized by G-CSF in the same fashion as CD34+ cells.
2. Both populations spontaneously leave bone marrow niches and are able to migrate.
3. Comparable self-renewal potential.
4. Comparable long-term multilineage engraftment.

Functional distinction between CD34+ and CD34- populations is based on the following:

1. CD34-negative populations (CD38+ and CD38-) have very low hematopoietic activity in vitro (colony assay). Because CD34-negative cells didn’t give any colonies in standard colony assay, this population was ignored as potential source of HSC.
2. In sharp contrast to CD34+ cells, CD34-negative HSC easily undergo ex vivo expansion resulting in increase SCID-repopulating potential. As soon as Lin-/CD34- cells acquired CD34 expression in culture, CD34+ were not able to repopulate mice, unlike fresh CD34+ cells.
3. Trend toward lymphoid-biased engraftment (Bhatia M. et al, 1998) after intravenous administration, but not biased multilineage engraftemnt after intra-bone injection (Sonoda group).
4. Lin-/CD34- cells have an impaired ability to home to bone marrow after intravenous injection due low level of CXCR4 expression (Sonoda reviewed 2008). These cells also have very poor SDF-1-mediated migration ability in vitro.

Markers and hierarchy
One of the main problem for isolation of HSC from CD34-negative population is the absence of known positive markers. It was shown that the most primitive Lin-/CD34- HSC are also negative for: CD38, HLA-DR, Flt3 and c-Kit (Bhatia, 1998, Sonoda 2003-2008).
As candidates for further isolation of HSC within CD34-negative population, two markers expressed on Lin-/CD34- cells were studied: ALDH and CD133. Unfortunately xenogeneic transplantation assays were not performed in both studies.

It could be very interesting to look at lineage repopulation by CD34-/ALDH+ and CD34-/CD133+ in sensitive xenotransplantation assays.

All groups showed that CD34-negative HSC are precursors for CD34+. Therefore CD34-negative cells are the most primitive human HSC in adult bone marrow and cord blood. Interestingly, there is a reversibility of CD34 expression on primitive HSC, at least for bone marrow.

Clinical relevance and perspectives
Very low clonogenic capacity in vitro and undiscovered markers which could allow positive selection are the main obstacles for clinical application and assessment of CD34-negative HSC. Some thoughts about CD34-negative HSC that could be significant for clinical use, I summarized as the following:
1. In order to assess clinical relevance of CD34-negative HSC, some positive markers should be discovered and new sensitive assays should be developed. For example, standard colony assay can estimate potency only CD34+ cells, but not CD34-negative.
2. In clinical HSC transplantation CD34-negative population can not be ignored, because it possesses high HSC activity and therapeutic potential.
3. Clinical significance of CD34-negative HSC supported by the fact that clinical outcome sometimes is not correlated with transplanted CD34+ cells quantity and quality.
4. In order to investigate therapeutic potential CD34-negative HSC, one should be brave enough to transplant one CD34-depleted cord blood unit with one fresh or ex vivo expanded and keep track of long-term engraftment. Another scenario – Lineage-negative cells versus CD34+ positively selected, whole bone marrow versus CD34+ selected. The former approach is currently under clinical trials for different conditions.
5. CD34-negative population could be more interesting target for clinical-grade HSC expansion compared to CD34+ cells.
6. CD34-negative population could be a candidate for leukemic stem cells and therefore could have prognostic and therapeutic value in leukemia clinic.

Connotea tag: CD34-negative

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