Hematopoietic stem cell enrichment in graft engineering

Enrich CD34+ hematopoietic stem cells (HSCs) and passively deplete unwanted cells for autologous and allogeneic hematopoietic stem cell transplantation (HSCT). CliniMACS Prodigy® and CliniMACS® Plus generate CD34+ stem cell products of up to 95% purity and with up to a 4.5 log10 depletion of T cells. The enriched product is ideal for HSCT or further engineering.1

The effective and concurrent passive depletion of T cells during CD34+ enrichment on CliniMACS Prodigy and CliniMACS Plus renders a highly pure population of HSCs that are used as stem cell grafts in various transplantation scenarios and settings. The following are examples of current applications.

1. Graft-versus-host disease (GVHD) prophylaxis

Enriched CD34+ cells were used as a GVHD prevention strategy in HLA-identical allogeneic HSCT to treat adult acute myeloid leukemia (AML) in first complete remission.2 Adding memory T cells to the graft supports immune reconstitution and significantly improves non-relapse mortality compared to pure CD34+ stem cell grafts while maintaining excellent GVHD prevention.3,4

2. Stem cell boost – rescue option for poor graft function (PGF)

Up to a quarter of allogeneic HSCT cases are complicated by infections, bleeding, and organ failure due to PGF.5 A boost of enriched CD34+ stem cells have shown rapid and sustained recovery with low rates of GVHD, plus improved overall survival rates after 3 years 6–12 and improved immune reconstitution.13 Researchers are also exploring stem cell boost to prevent PGF in other transplantation contexts14,15 and after CAR T cell therapy16,17.

3. The haplo-cord approach

Combined haplo-cord HSCT uses CD34+ stem cells to bridge the gap between transplant and primary engraftment of a cord blood graft. The haploidentical CD34+-enriched cells are transplanted concurrently with cord blood grafts and have led to sustained primary engraftment even with cord blood units containing very low stem cell numbers.18–20

4. CD34+ cell enrichment in autologous HSCT

CD34+ cell-enriched grafts are also used for HSCT in some early childhood tumors21,22, non-Hodgkin lymphoma and other diseases in adults23,24. The technique is also used to deplete autoreactive cells from HSC grafts for autoimmune diseases like systemic lupus erythematosus and systemic sclerosis.25–27

CliniMACS® Technology in stem cell transplantation 

Discover the CliniMACS Systems for GMP-compliant cell depletion and enrichment strategies that help clinicians overcome graft-versus-host disease, infections, and poor graft function.

Workflow overview of the CD34 enrichment process on the CliniMACS Prodigy and the CliniMACS Plus Instrument.

HSCs in starting material collected by leukapheresis are first labeled and then captured via magnetic cell separation while unlabeled cells flow through the separation column. The enriched CD34+ cells are then collected in a target cell bag. 

Automation solutions

Whether complete end-to-end cell manufacturing or just cell enrichment and depletion, the CliniMACS Prodigy and CliniMACS Plus automate clinical-scale processes.

How to enrich CD34+ hematopoietic stem cells from blood products using CliniMACS Technology? 

The automated and closed process of the CliniMACS Prodigy and CliniMACS Plus enables sterile, reproducible and GMP-compliant cell enrichment

Dedicated product portfolio

CliniMACS CD34 enrichment is widely used to generate pure CD34+ cell populations from HSC grafts. The resulting products are passively depleted of T cells and widely used in strategies for various approaches in allogeneic stem cell transplantation and HSC engineering. 

In addition to their use as highly pure graft material for HSCT, enriched CD34+ HSCs are a starting point for further cell processing. The HSCs are used in the manufacture of cells modified via gene engineering to treat specific blood disorders.28  Alternatively, the graft material can be actively adapted by removing unwanted cell populations and retaining cells that support therapeutic goals.

Manufacturing gene-engineered HSCs

Viral transduction enables altering CD34+ HSCs to treat inherited disorders that affect the constitution or performance of blood, such as sickle cell anemia or primary immunodeficiencies.29–31 In a first step, CD34+ HSCs are enriched on the CliniMACS Prodigy or CliniMACS Plus. Subsequent transduction and harvesting of engineered HSCs are then conducted on the CliniMACS Prodigy.

Precise graft engineering via active T cell depletion

The targeted removal of unwanted cells is a form of graft engineering that retains therapeutically beneficial cell combinations within a graft. Well-defined populations of T cells are actively depleted and, depending on the depletion strategy, the resulting graft contains HSCs, other progenitor cells, natural killer (NK) cells, monocytes, dendritic cells, and desirable T cells that facilitate engraftment and may help to prevent GVHD.

1. Hümmer, C. et al. (2016) Automation of cellular therapy product manufacturing: results of a split validation comparing CD34 selection of peripheral blood stem cell apheresis product with a semi-manual vs. an automatic procedure. J. Transl. Med. 14: 76–83.

2. Pasquini, M. C. et al. (2012) Comparative outcomes of donor graft CD34+ selection and immune suppressive therapy as graft-versus-host disease prophylaxis for patients with acute myeloid leukemia in complete remission undergoing HLA-matched sibling allogeneic hematopoietic cell transplantation. J. Clin. Oncol. 30: 3194–3201.

3. Luznik, L. et al. (2022) Randomized phase III BMT CTN trial of calcineurin inhibitor-free chronic graft-versus-host disease interventions in myeloablative hematopoietic cell transplantation for hematologic malignancies. J. Clin. Oncol. 40: 356–368.

4. Bleakley, M. et al. (2022) Naive T-cell depletion to prevent chronic graft-versus-host disease. J. Clin. Oncol. JCO2101755. Epub ahead of print, Jan. 10.

5. Lee, K. H. et al. (2004) Failure of trilineage blood cell reconstitution after initial neutrophil engraftment in patients undergoing allogeneic hematopoietic cell transplantation – Frequency and outcomes. Bone Marrow Transplant. 33: 729–734.

6. Ghobadi, A. et al. (2017) Fresh or cryopreserved CD34+-selected mobilized peripheral blood stem and progenitor cells for the treatment of poor graft function after allogeneic hematopoietic cell transplantation. Biol. Blood Marrow Transplant. 23: 1072–1077.

7. Stasia, A. et al. (2014) CD34 selected cells for the treatment of poor graft function after allogeneic stem cell transplantation. Biol. Blood Marrow Transplant. 20: 1440–1443.

8. Klyuchnikov, E. et al. (2014) CD34+-selected stem cell boost without further conditioning for poor graft function after allogeneic stem cell transplantation in patients with hematological malignancies. Biol. Blood Marrow Transplant. 20: 382–386.

9. Haen, S.P. et al. (2015) Poor graft function can be durably and safely improved by CD34+-selected stem cell boosts after allogeneic unrelated matched or mismatched hematopoietic cell transplantation. J. Cancer Res. Clin. Oncol. 141: 2241–2251.

10. Askaa, B. et al. (2014) Treatment of poor graft function after allogeneic hematopoietic cell transplantation with a booster of CD34-selected cells infused without conditioning. Bone Marrow Transplant. 49: 720–721.

11. Chandra, S. et al. (2018) Post-transplant CD34+ selected stem cell “boost” for mixed chimerism after reduced-intensity conditioning hematopoietic stem cell transplantation in children and young adults with primary immune deficiencies. Biol. Blood Marrow Transplant. 24: 1527–1529.

12. Mohty, R. et al. (2019) CD34+-selected stem cell “boost” for poor graft function after allogeneic hematopoietic stem cell transplantation. Curr. Res. Transl. Med. 67: 112–114.

13. Cuadrado, M. et al. (2020) Predictors of recovery following allogeneic CD34+-selected cell infusion without conditioning to correct poor graft function. Haematologica 105: 2639–2646.

14. Mainardi, C. et al. (2018) CD34+ selected stem cell boosts can improve poor graft function after paediatric allogeneic stem cell transplantation. Br. J. Haematol. 180: 90–99.

15. Abboud, R. et al. (2021) Can planned CD34+ stem cell boost prevent poor graft function after peripheral blood haploidentical hematopoietic transplantation? Leuk. Lymphoma 62: 749–751.

16. Mullanfiroze, K. et al. (2022) CD34+-selected stem cell boost can safely improve cytopenias following CAR T-cell therapy. Blood Adv. 6: 4715–4718.

17. Rejeski, K. et al. (2022) Safety and feasibility of stem cell boost as a salvage therapy for severe hematotoxicity after CD19 CAR T-cell therapy. Blood Adv. 6: 4719–4725.

18. Liu, H. and van Besien, K. (2015) Alternative donor transplantation – “mixing and matching”: the role of combined cord blood and haplo-identical donor transplantation (haplo-cord SCT) as a treatment strategy for patients lacking standard donors? Curr. Hematol. Malig. Rep. 10: 1–7.

19. Kwon, M. et al. (2014) Haplo-cord transplantation using CD34+ cells from a third-party donor to speed engraftment in high-risk patients with hematologic disorders. Biol. Blood Marrow Transplant. 20: 2015–2022.

20. Van Besien, K. et al. (2019) Haploidentical vs haplo-cord transplant in adults under 60 years receiving fludarabine and melphalan conditioning. Blood Adv. 3: 1858–1867.

21. Ballova, V. et al. (2008) Autologous stem cell transplantation with selected CD34+ cells and unmanipulated peripheral blood stem cells in patients with relapsed and refractory Hodgkin’s lymphoma: a single centre experience. Neoplasma 55: 428–436.

22. Marabelle, A. et al. (2011) CD34+ immunoselection of autologous grafts for the treatment of high-risk neuroblastoma. Pediatr. Blood Cancer 56: 134–142.

23. Yahng, S. A. et al. (2014) Influence of ex vivo purging with CliniMACS CD34(+) selection on outcome after autologous stem cell transplantation in non-Hodgkin lymphoma. Br. J. Haematol. 164: 555–564.

24. Berger, M. D. et al. (2015) CD34+ selected versus unselected autologous stem cell transplantation in patients with advanced-stage mantle cell and diffuse large B-cell lymphoma. Leuk. Res. 39: 561–567.

25. Alchi, B. et al. (2013) Autologous haematopoietic stem cell transplantation for systemic lupus erythematosus: data from the European Group for Blood and Marrow Transplantation registry. Lupus 22: 245–253.

26. Henes, J. C. et al. (2012) Optimization of autologous stem cell transplantation for systemic sclerosis — a single-center long-term experience in 26 Patients with severe organ manifestations. J. Rheumatol. 39: 269–275.

27. van Laar, J. M. et al. (2014) Autologous hematopoietic stem cell transplantation vs intravenous pulse cyclophosphamide in diffuse cutaneous systemic sclerosis. A randomized clinical trial. JAMA 311: 2490–2498.

28. Frangoul, H. et al. (2021) CRISPR-Cas9 Gene Editing for Sickle Cell Disease and beta-Thalassemia. N. Engl. J. Med. 384: 252–260.

29. de Dreuzy E. et al. (2016) Current and future alternative therapies for beta-thalassemia major. Biomed. J. 39: 24–38.

30. Lebensburger, J. and Persons D.A. (2008) Progress toward safe and effective gene therapy for beta-thalassemia and sickle cell disease. Curr. Opin. Drug Discov. Devel. 11: 225–232.

31. Papanikolaou E. and Anagnou N.P. (2010) Major challenges for gene therapy of thalassemia and sickle cell disease. Curr. Gene Ther. 10: 404–412.

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