Characterization of Retroviral Gene Transfer into Highly Purified Human CD34− Cells with Primitive Hematopoietic Capacity
Bibliographic record
Abstract
Primitive human hematopoietic cells have recently been identified within a rare subfraction of CD34− lineage-depleted (Lin−) cells and further characterized by their restriction to a rarer subset expressing AC133. Here we show that CD34−AC133+Lin− cells can be transduced by retrovirus at a comparatively higher efficiency than either CD34−AC133−Lin− or CD34+CD38−Lin− cells. Subpopulations were transduced by enhanced green fluorescent protein (eGFP)-containing retrovirus in serum-free conditions. During the culture period, both CD34−AC133+Lin− and CD34+CD38−Lin− subfractions expanded, whereas CD34−AC133−Lin− cells could not be sustained. Fluorescent microscopic examination of progenitors assayed by colony-forming units (CFU) derived from CD34−AC133+Lin− cells revealed expression of eGFP, with the presence of provirus confirmed by clonal PCR analysis. Flow cytometry detecting eGFP revealed that cultures seeded with CD34−AC133+Lin− cells had a greater than threefold higher frequency of eGFP+ cells compared with transduced cultures of CD34+CD38−Lin− cells. Our results demonstrate that retroviral transduction efficiency and level of transgene expression into CD34−AC133+Lin− cells is distinct to either CD34−AC133−Lin− or CD34+CD38−Lin− cells. This study represents the first evaluation of retroviral transduction into this population of primitive CD34− cells, and therefore provides the basis for optimization of gene transfer protocols to examine the role of gene-marked CD34− stem cells in a clinical setting. Primitive human hematopoietic cells have recently been identified within a rare subfraction of CD34− lineage-depleted (Lin−) cells and further characterized by their restriction to a rarer subset expressing AC133. Here we show that CD34−AC133+Lin− cells can be transduced by retrovirus at a comparatively higher efficiency than either CD34−AC133−Lin− or CD34+CD38−Lin− cells. Subpopulations were transduced by enhanced green fluorescent protein (eGFP)-containing retrovirus in serum-free conditions. During the culture period, both CD34−AC133+Lin− and CD34+CD38−Lin− subfractions expanded, whereas CD34−AC133−Lin− cells could not be sustained. Fluorescent microscopic examination of progenitors assayed by colony-forming units (CFU) derived from CD34−AC133+Lin− cells revealed expression of eGFP, with the presence of provirus confirmed by clonal PCR analysis. Flow cytometry detecting eGFP revealed that cultures seeded with CD34−AC133+Lin− cells had a greater than threefold higher frequency of eGFP+ cells compared with transduced cultures of CD34+CD38−Lin− cells. Our results demonstrate that retroviral transduction efficiency and level of transgene expression into CD34−AC133+Lin− cells is distinct to either CD34−AC133−Lin− or CD34+CD38−Lin− cells. This study represents the first evaluation of retroviral transduction into this population of primitive CD34− cells, and therefore provides the basis for optimization of gene transfer protocols to examine the role of gene-marked CD34− stem cells in a clinical setting.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".