Title page for ETD etd-1205102-085840

Document Type thesis

Author Name Chomal, Manish R

Email Address chomalmanish at yahoo.com

URN etd-1205102-085840

Title Analysis of Telomerase Activity and Telomere Lengths in Human Umbilical Cord Cell Populations During Ex Vivo Amplification of Hematopoietic Stem Cells

Degree MS

Department Biology & Biotechnology

Advisors
Dr. David Adams, Advisor
Dr. Jill Rulfs, Committee Member
Dr. Daniel Gibson, Committee Member

Keywords
Telomerase Activity
Telomere Lengths
Hematopoietic Stem Cells

Date of Presentation/Defense 2002-12-02

Availability unrestricted

Abstract
Human umbilical cord blood (CB) hematopoietic stem cells (HSCs) have well
established applications for cellular therapy. Current protocols for isolating HSCs from
bone marrow or cord select for CD34 + cells, however some CD34 - populations have
recently been shown to also contain strong HSC activity. Thus the positive selection of
HSCs based on cell surface markers remains controversial. However, it is clear from the
literature that differentiated hematopoietic cells (lineage positive, Lin + ), representing the
vast majority (>90%) of most blood populations, contain no long-term reconstitution
potential. Thus Viacell Inc. (Worcester, MA) expands and enriches its populations of
cells containing HSCs by removing only those Lin + cells known not to contain HSCs.
This is accomplished on two separation columns (post-sep-1, and post-sep-2) (separated
by 7 days of cell growth) that contain a variety of antibodies to known differentiation
surface markers. Although this process strongly enriches functional HCSs, these
primitive cell populations remain biochemically uncharacterized.
Because HSC populations containing long chromosomal telomeres and high
telomerase activity (which helps maintain telomeres) have been shown to display the
strongest long-term reconstitution potential, the purpose of this thesis was to investigate
these two parameters in selected samples of Viacell’s ex vivo amplification procedure.
Two specific hypotheses were tested: 1. the removal of Lin + cells will appear to increase
the telomerase activity and telomere lengths in the remaining cell population, and 2. these
two parameters will decrease upon hematopoietic cell differentiation and proliferation.
Telomerase activity was assayed using a telomeric repeat amplication protocol (TRAP),
and normalized relative to a cancer cell line positive control. Relative to fresh cord blood, telomerase activity was found to increase significantly in post-sep-1 (from 8.5 ±
1.5% to 76.2 ± 4.9%, p = 0.0001, n = 5) and post-sep-2 (8.5 ± 1.5% to 111.3 ± 4.9%, p =
0.0001, n = 5) fractions following the removal of Lin + cells. This increase was found to
be highly reproducible, showing very low intra-cord and inter-cord variability. Telomere
lengths were assayed using a telomere length assay (TLA). Relative to fresh cord blood,
telomere lengths increased significantly in post-sep-1 (from 10 to 12 kb, n = 2) and post-sep-
2 (from 10 to 14 kb, p = 0.001, n = 2) fractions. These apparent increases likely
result from the direct removal of cells low in telomerase activity with short telomeres
since the Lin + cells from the post-sep-1 column were found to contain relatively low
telomerase activity (32.1 ± 15%, p = 0.001, n = 2) and short telomeres (7.5 kb, p =
0.001), which supports our first hypothesis. Finally, we show that telomerase activity
and telomere lengths decreased in Day-14 cells (expanded and differentiated 14 days)
relative to post-sep-2 (from 111.8 ± 19.6% to 54 ± 21.2%, p = 0.001, n = 3 for the TRAP,
and from 14 kb to 9 kb, p = 0.0001, n = 2 for the TLA). Those two parameters also
decreased in pre-sep-3 cells (terminally differentiated by treatment with All Trans
Retinoic Acid for 14 days) relative to post-sep-2 (from 111.3 ± 4.9% to 14.8 ± 1.7%, p =
0.0001, n = 6 for the TRAP, and from 14 kb to 7.5 kb, p = 0.001 for the TLA), supporting
our second hypothesis. Telomerase activity was found to not directly correlate with
CD34 + CD38 - content, supporting recent observations that a significant portion of HSCs
reside outside this population.

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Document Type	thesis
Author Name	Chomal, Manish R
Email Address	chomalmanish at yahoo.com
URN	etd-1205102-085840
Title	Analysis of Telomerase Activity and Telomere Lengths in Human Umbilical Cord Cell Populations During Ex Vivo Amplification of Hematopoietic Stem Cells
Degree	MS
Department	Biology & Biotechnology
Advisors	Dr. David Adams, Advisor Dr. Jill Rulfs, Committee Member Dr. Daniel Gibson, Committee Member
Keywords	Telomerase Activity Telomere Lengths Hematopoietic Stem Cells
Date of Presentation/Defense	2002-12-02
Availability	unrestricted
Abstract Human umbilical cord blood (CB) hematopoietic stem cells (HSCs) have well established applications for cellular therapy. Current protocols for isolating HSCs from bone marrow or cord select for CD34 + cells, however some CD34 - populations have recently been shown to also contain strong HSC activity. Thus the positive selection of HSCs based on cell surface markers remains controversial. However, it is clear from the literature that differentiated hematopoietic cells (lineage positive, Lin + ), representing the vast majority (>90%) of most blood populations, contain no long-term reconstitution potential. Thus Viacell Inc. (Worcester, MA) expands and enriches its populations of cells containing HSCs by removing only those Lin + cells known not to contain HSCs. This is accomplished on two separation columns (post-sep-1, and post-sep-2) (separated by 7 days of cell growth) that contain a variety of antibodies to known differentiation surface markers. Although this process strongly enriches functional HCSs, these primitive cell populations remain biochemically uncharacterized. Because HSC populations containing long chromosomal telomeres and high telomerase activity (which helps maintain telomeres) have been shown to display the strongest long-term reconstitution potential, the purpose of this thesis was to investigate these two parameters in selected samples of Viacell’s ex vivo amplification procedure. Two specific hypotheses were tested: 1. the removal of Lin + cells will appear to increase the telomerase activity and telomere lengths in the remaining cell population, and 2. these two parameters will decrease upon hematopoietic cell differentiation and proliferation. Telomerase activity was assayed using a telomeric repeat amplication protocol (TRAP), and normalized relative to a cancer cell line positive control. Relative to fresh cord blood, telomerase activity was found to increase significantly in post-sep-1 (from 8.5 ± 1.5% to 76.2 ± 4.9%, p = 0.0001, n = 5) and post-sep-2 (8.5 ± 1.5% to 111.3 ± 4.9%, p = 0.0001, n = 5) fractions following the removal of Lin + cells. This increase was found to be highly reproducible, showing very low intra-cord and inter-cord variability. Telomere lengths were assayed using a telomere length assay (TLA). Relative to fresh cord blood, telomere lengths increased significantly in post-sep-1 (from 10 to 12 kb, n = 2) and post-sep- 2 (from 10 to 14 kb, p = 0.001, n = 2) fractions. These apparent increases likely result from the direct removal of cells low in telomerase activity with short telomeres since the Lin + cells from the post-sep-1 column were found to contain relatively low telomerase activity (32.1 ± 15%, p = 0.001, n = 2) and short telomeres (7.5 kb, p = 0.001), which supports our first hypothesis. Finally, we show that telomerase activity and telomere lengths decreased in Day-14 cells (expanded and differentiated 14 days) relative to post-sep-2 (from 111.8 ± 19.6% to 54 ± 21.2%, p = 0.001, n = 3 for the TRAP, and from 14 kb to 9 kb, p = 0.0001, n = 2 for the TLA). Those two parameters also decreased in pre-sep-3 cells (terminally differentiated by treatment with All Trans Retinoic Acid for 14 days) relative to post-sep-2 (from 111.3 ± 4.9% to 14.8 ± 1.7%, p = 0.0001, n = 6 for the TRAP, and from 14 kb to 7.5 kb, p = 0.001 for the TLA), supporting our second hypothesis. Telomerase activity was found to not directly correlate with CD34 + CD38 - content, supporting recent observations that a significant portion of HSCs reside outside this population.
Files	chomal.pdf