Human hematopoietic stem cells from mobilized peripheral blood can be purified based on CD49f integrin expression (2024)

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CORRESPONDENCE| September 24, 2015

Heather D. Huntsman,

Heather D. Huntsman *

1National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Taha Bat,

Taha Bat *

2National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Hai Cheng,

Hai Cheng

3National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

4Department of Hematology, Affiliated Hospital of Xuzhou Medical College, Xuzhou, Jiangsu Province, China

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Ayla Cash,

Ayla Cash

5National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Patali S. Cheruku,

Patali S. Cheruku

6National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Jian-Fei Fu,

Jian-Fei Fu

7National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

8Shanghai Institute of Hematology, Rui-jin Hospital, Shanghai Jiao-Tong University, Shanghai, China

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Keyvan Keyvanfar,

Keyvan Keyvanfar

9National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Richard W. Childs,

Richard W. Childs

10National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Cynthia E. Dunbar,

Cynthia E. Dunbar

11National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Andre Larochelle

Andre Larochelle

12National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD

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Blood (2015) 126 (13): 1631–1633.

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Heather D. Huntsman, Taha Bat, Hai Cheng, Ayla Cash, Patali S. Cheruku, Jian-Fei Fu, Keyvan Keyvanfar, Richard W. Childs, Cynthia E. Dunbar, Andre Larochelle; Human hematopoietic stem cells from mobilized peripheral blood can be purified based on CD49f integrin expression. Blood 2015; 126 (13): 1631–1633. doi: https://doi.org/10.1182/blood-2015-07-660670

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Subjects:

Hematopoiesis and Stem Cells

Topics:

hematopoietic stem cells, integrin alpha6, integrins

To the editor:

Granulocyte colony-stimulating factor (G-CSF)–mobilized peripheral blood (MPB) is the preferred source of hematopoietic stem cells (HSCs) and has outcompeted bone marrow for HSC-based therapies.1 Umbilical cord blood (UCB) represents an alternative source of HSCs commonly used in preclinical investigations, but limited cell dose has precluded its wide clinical utility.2 Purification of HSCs to hom*ogeneity from these various sources is essential to better understand their unique biology and identify novel therapeutic strategies targeting these cells. In humans, the CD34+CD38CD45RACD90+ phenotype is now customarily accepted to define a cell population highly enriched in HSCs.3 Recently, Notta et al4 further refined this phenotype with the addition of CD49f, an integrin known to mediate HSC niche interactions.5 Using UCB, they estimated that 1:10 cells within the CD34+CD38CD45RACD90+CD49f+ population had long-term repopulating activity in vivo and could be distinguished from transiently engrafting multipotent progenitors (MPPs) defined as CD34+CD38CD45RACD90CD49f. When these highly purified UCB HSCs were further sorted based on high efflux of the mitochondrial dye rhodamine 123 (Rholo),6 an additional twofold HSC enrichment was obtained and, strikingly, a fraction of murine recipients transplanted with single-sorted CD34+CD38CD45RACD90+CD49f+Rholo cells displayed multilineage chimerism. Hence, this report was the first to demonstrate purification of human HSCs at single-cell resolution from UCB. In this study, we investigated whether this panel of 6 markers established in UCB could also mark HSCs in MPB.

Leukapheresis was performed on 14 healthy volunteers following 5 days of G-CSF mobilization. CD34+ cells were enriched from the mononuclear cell (MNC) concentrates. Details of the experimental approach can be found in supplemental Methods (available on the Blood Web site). We first investigated whether markers used to enrich for HSCs and MPPs in UCB could also be used to phenotypically identify these 2 subsets in MPB cells. The average frequency of CD34+ cells within the G-CSF–MPB MNC concentrates was 1 in 100 cells (1.05% ± 0.10%; Figure 1A) with purity ≥98.8%. Using stringent gates (Figure 1B), we found that, on average, 1 in 145 000 MPB MNCs (0.00069% ± 0.00008%) had an HSC phenotype, and 1 in 17 000 MPB MNCs were phenotypically defined as MPPs (0.0059% ± 0.0007%) (Figure 1A). Similar results were obtained in MPB MNCs derived from the phylogenetically related rhesus macaque (supplemental Figure 1A-B) and from 6 healthy subjects mobilized with plerixafor (supplemental Figure 1C-D). Overall, these data indicate a highly consistent pattern of expression of these markers in various sources of MPB cells.

Figure 1

Human hematopoietic stem cells from mobilized peripheral blood can be purified based on CD49f integrin expression (1)

View largeDownload PPT

Phenotypic analysis and engraftment potential of human G-CSF–MPB. Phenotypic analysis of G-CSF–MPB MNCs (A-B). (A) Frequency of human CD34+ cells and phenotypically defined MPPs (CD34+CD38CD45RACD90CD49f, abbreviated CD49f) and HSCs (CD34+CD38CD45RACD90+CD49f+Rholo, abbreviated CD49f+Rholo) in G-CSF-MPB MNCs obtained from 14 healthy subjects. (B) Representative flow cytometry analysis for the detection of phenotypically defined MPPs and HSCs in G-CSF-MPB MNCs; the frequency of each subpopulation is based on the parent gate shown on the top right of each plot. Using a limiting dilution transplantation approach, the frequency of HSC within the CD34+CD38CD45RACD90+CD49f+Rholo subfraction was determined (C-E). (C) Schematic of the experimental design. (D) Table outlining the number of mice transplanted at each dose, the number of mice that engrafted, and the percentage of mice that failed to engraft at each respective dose. (E) Semilogarithmic plot of the frequency of long-term repopulating cells within the CD34+CD38CD45RACD90+CD49f+Rholo subfraction measured by extreme limiting dilution analysis. (D) Representative flow cytometry analysis from a single NSG mouse recipient transplanted with 200 CD34+CD38CD45RACD90+CD49f+Rholo cells for human cell engraftment (CD45) and lineage contribution. Analysis is representative of 3 independent experiments (n = 7 mice).

Figure 1

Human hematopoietic stem cells from mobilized peripheral blood can be purified based on CD49f integrin expression (2)

View largeDownload PPT

Phenotypic analysis and engraftment potential of human G-CSF–MPB. Phenotypic analysis of G-CSF–MPB MNCs (A-B). (A) Frequency of human CD34+ cells and phenotypically defined MPPs (CD34+CD38CD45RACD90CD49f, abbreviated CD49f) and HSCs (CD34+CD38CD45RACD90+CD49f+Rholo, abbreviated CD49f+Rholo) in G-CSF-MPB MNCs obtained from 14 healthy subjects. (B) Representative flow cytometry analysis for the detection of phenotypically defined MPPs and HSCs in G-CSF-MPB MNCs; the frequency of each subpopulation is based on the parent gate shown on the top right of each plot. Using a limiting dilution transplantation approach, the frequency of HSC within the CD34+CD38CD45RACD90+CD49f+Rholo subfraction was determined (C-E). (C) Schematic of the experimental design. (D) Table outlining the number of mice transplanted at each dose, the number of mice that engrafted, and the percentage of mice that failed to engraft at each respective dose. (E) Semilogarithmic plot of the frequency of long-term repopulating cells within the CD34+CD38CD45RACD90+CD49f+Rholo subfraction measured by extreme limiting dilution analysis. (D) Representative flow cytometry analysis from a single NSG mouse recipient transplanted with 200 CD34+CD38CD45RACD90+CD49f+Rholo cells for human cell engraftment (CD45) and lineage contribution. Analysis is representative of 3 independent experiments (n = 7 mice).

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We next performed limiting dilution analysis7 to measure the frequency of HSCs within the CD34+CD38CD45RACD90+CD49f+Rholo population using G-CSF–MPB cells (Figure 1C). A total of 53 NSG mice were transplanted by intravenous route and engraftment was defined as ≥0.1% human CD45+ cells. Limiting dilution analysis indicates that 2.3% (1 in 43 cells; 95% confidence interval, 1 in 23.3 to 1 in 78.4 cells) of the G-CSF–MPB CD34+CD38CD45RACD90+CD49f+Rholo fraction has long-term repopulating activity in NSG mice (Figure 1D-E). One of 15 animals transplanted with a single cell displayed robust engraftment (9.2% human CD45+ cells; data not shown). Additionally, all major hematopoietic lineages could be detected in the bone marrow of engrafted mice (n = 7), including cells of the erythroid (2.9% ± 2.5%), myeloid (8.2% ± 4.2%), megakaryocytic (6.6% ± 2.7%), and B-lymphoid (75.4% ± 4.5%) lineages (Figure 1F).

In this study, we provide evidence that CD49f can be used in combination with common HSC markers to enrich cells with long-term engraftment potential from G-CSF-MPB samples. Detection of engraftment in one animal transplanted with a single cell indicates that human HSCs derived from G-CSF-MPB samples express CD49f. Because 1 in 145 000 G-CSF–MPB MNCs are CD34+CD38CD45RACD90+CD49f+Rholo (Figure 1A) and 2.3% of this fraction has repopulating activity (Figure 1D-E), the overall HSC frequency in G-CSF–MPB MNCs is estimated at 1 in 6.2 × 106 cells, consistent with a previously reported estimate using this model.8 These data contribute to the body of evidence indicating a five- to 10-fold lower HSC frequency in MPB compared with UCB sources.8 However, because the total number of CD34+ cells obtained after G-CSF mobilization of a healthy subject is on average 100- to 200-fold superior to CD34+ cell numbers obtained from a single UCB unit, MPB samples represent a more practical source of HSCs and MPPs for isolation of sufficient numbers of cells required for both clinical applications and fundamental investigations of the molecular determinants of these cell populations.

The online version of this article contains a data supplement.

Authorship

Acknowledgments: The authors thank David Stroncek and the National Institutes of Health Department of Transfusion Medicine and Cell Processing Section staff for apheresis, selection, and cryopreservation of human CD34+ cells; Roumiana Nenkova-Dimtcheva and the Hematology Branch outpatient clinic nursing staff for providing G-CSF administration teaching to healthy subjects; and Crystal Thomas and the mouse core facility staff, as well as Robert E. Donahue and the nonhuman primate core facility staff, for excellent animal care. This work was supported by the intramural research program of the National Heart, Lung and Blood Institute of the National Institutes of Health.

Contribution: H.D.H., T.B., and A.L. designed and performed the experimental procedures, analyzed the data, and wrote and edited the manuscript; H.C., A.C., P.S.C., and J.-F.F. performed some of the flow stains and analyses; K.K. sorted the cell populations; R.W.C. provided the AMD3100-mobilized samples; and C.E.D. edited the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Andre Larochelle, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892; e-mail: larochea@nhlbi.nih.gov.

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Author notes

*

H.D.H. and T.B. contributed equally to this study.

2015

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September 24 2015

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Human hematopoietic stem cells from mobilized peripheral blood can be purified based on CD49f integrin expression (2024)

FAQs

How many CD34 cells do you need to collect for stem cell mobilization to be considered a success? ›

The minimum required count of PBSC for ASCT is 2 × 106 CD34+ cells per kilogram of body weight. [4] Mobilization strategies typically involve the use of cytokines alone or in combination with chemotherapeutic agents.

Can hematopoietic stem cells be harvested from the blood? ›

The most common way to harvest stem cells involves temporarily removing blood from the body, separating out the stem cells, and then returning the blood to the body.

What is mobilization of peripheral blood stem cells? ›

Stem cell mobilization is a process whereby stem cells are stimulated out of the bone marrow space (eg, the hip bones and the chest bone) into the bloodstream so they are available for collection for future reinfusion. The cells are then preserved, frozen, and stored until the time of transplant.

What is the transplantation of hematopoietic stem cells from the peripheral blood? ›

A procedure in which a patient receives healthy blood-forming cells (stem cells) to replace their own stem cells that have been destroyed by disease or by the radiation or high doses of anticancer drugs that are given as part of the procedure.

What is the optimum number of CD34+ peripheral blood stem cells for an autologous transplant? ›

Background/aim. High-dose melphalan and autologous hematopoietic stem cell transplantation (AHSCT) is the standard treatment strategy for multiple myeloma (MM) patients who are eligible for it. The recommended dose of CD34+ hematopoietic progenitor cells (HPCs) for adequate engraftment is above 2 × 106/kg.

What percentage of CD34 cells are in peripheral blood? ›

There is a consensus that, in normal individuals, the percentage of CD34+ cells in the peripheral blood is very low, ranging from 0.01% to 0.1%, or an absolute count of 0.6 to 6 CD34+ cells/µL in a patient with a WBC count of 6,000 for example.

Can stem cells be collected from peripheral blood? ›

Peripheral blood stem cell (PBSC) donation is a nonsurgical procedure to collect blood-forming cells for bone marrow transplants. Peripheral blood stem cell (PBSC) donation is one of two methods of collecting blood-forming cells for bone marrow transplants.

Can stem cells regenerate peripheral nerves? ›

In addition, neural stem cells also promote regeneration of the axonal myelin sheath, angiogenesis, and immune regulation. It can be concluded that neural stem cells promote the repair of peripheral nerve injury through a variety of ways.

What is the peripheral blood source of hematopoietic stem cells? ›

Hematopoietic stem cells (HSCs) are multipotent primitive cells that can develop into all types of blood cells, including myeloid-lineage and lymphoid-lineage cells (1). HSCs can be found in several organs, such as peripheral blood (PB), bone marrow (BM), and umbilical cord blood (UCB).

What is the life expectancy after stem cell transplant? ›

Results. Median follow-up was 9 years, and 3,788 patients had been observed for 10 or more years. The probability of being alive 10 years after HCT was 85%. The chief risk factors for late death included older age and chronic graft-versus-host disease (GVHD).

What is the success rate of peripheral blood stem cell transplant? ›

Bone marrow or peripheral blood stem cell transplantation from a histocompatible sibling produces long-term survival rates of 75% to 80%.

What is the survival rate for hematopoietic stem cell transplant? ›

Median survival after ICU admission was 50.5 (CI 95%, 20–430) days for allogeneic HSCT recipients and 1115 (CI 95%, 337–NA) days for autologous HSCT recipients. At 1 year after ICU admission, 21 (36.8%) autologous HSCT recipients and 18 (69.2%) allogeneic HSCT recipients had died (Figure 4).

What is a good CD34 count? ›

While the minimum number of hematopoietic stem cells for a successful transplantation has been established, there is no definite information on the optimum number or the maximum number of stem cells. It is generally agreed that a minimum dose of 2.5 x 10(6) CD34+ cells is necessary for successful engraftment.

How many stem cells are needed for a stem cell transplant? ›

What are the steps in an autologous stem cell transplant? Collecting the stem cells: Before treatment begins, a needle is inserted into the patient's arm vein. Blood is withdrawn and redirected into a special machine that removes about 4 million stem cells – the amount needed for a transplant.

How do you calculate CD34 count? ›

The predicted number of collected cells was calculated using the following formula: Total number of CD34+ (cells×106/kg) yield=[(peripheral CD34+ cells/μL)×(0.43)/recipient body weight (kg)]×total liters processed.

How many cells are needed for cell therapy? ›

The summary is that almost all current T-cell therapy doses are delivered as follows: Total dose in the range of 3 × 106 to 3 × 107 cells/kg. Delivered in escalating doses starting as low as 106 cells and progressing to target doses of 109 or 1010 cells.

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