Techniques to isolate haematopoietic stem cells

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Since haematopoietic stem cells cannot be isolated as a pure population, it is not possible to identify them under a microscope.[ citation needed ] Therefore, there are many techniques to isolate haematopoietic stem cells (HSCs). HSCs can be identified or isolated by the use of flow cytometry where the combination of several different cell surface markers is used to separate the rare HSCs from the surrounding blood cells. HSCs lack expression of mature blood cell markers and are thus, called Lin-. Lack of expression of lineage markers is used in combination with detection of several positive cell-surface markers to isolate HSCs. In addition, HSCs are characterized by their small size and low staining with vital dyes such as rhodamine 123 (rhodamine lo) or Hoechst 33342 (side population).

Contents

CD34+ Cells can be isolated by 4 different techniques from peripheral blood samples

  1. By magnetic beads with MACS
  2. By FACS
  3. By labelled anti-antibodies
  4. Manually by culture. Since CD34 are in suspension culture and almost all cells in PBMC gets adhered, CD34 can be isolated through this process

Cluster of differentiation and other markers

The classical marker of human HSC is CD34 first described independently by Civin et al. and Tindle et al. [1] [2] [3] [4] It is used to isolate HSC for reconstitution of patients who are haematologically incompetent as a result of chemotherapy or disease.

Many markers belong to the cluster of differentiation series, like: CD34, CD38, CD90, CD133, CD105, CD45, and also c-kit – the receptor for stem cell factor.

There are many differences between the human and murine hematopoietic cell markers for the commonly accepted type of hematopoietic stem cells. [5]

However, not all stem cells are covered by these combinations that, nonetheless, have become popular. In fact, even in humans, there are hematopoietic stem cells that are CD34 −/CD38 −. [6] [7] Also some later studies suggested that earliest stem cells may lack c-kit on the cell surface. [8] For human HSCs use of CD133 was one step ahead as both CD34 + and CD34 − HSCs were CD133 +.

Traditional purification method used to yield a reasonable purity level of mouse hematopoietic stem cells, in general, requires a large (~10–12) battery of markers, most of which were surrogate markers with little functional significance, and thus partial overlap with the stem cell populations and sometimes other closely related cells that are not stem cells. Also, some of these markers (e.g., Thy1) are not conserved across mouse species, and use of markers like CD34 − for HSC purification requires mice to be at least 8 weeks old.

SLAM code

Alternative methods that could give rise to a similar or better harvest of stem cells is an active area of research, and are presently[ when? ] emerging. One such method uses a signature of SLAM family cell surface molecules. The SLAM (Signaling lymphocyte activation molecule) family is a group of more than 10 molecules whose genes are located mostly tandemly in a single locus on chromosome 1 (mouse), all belonging to a subset of the immunoglobulin gene superfamily, and originally thought to be involved in T-cell stimulation. This family includes CD48, CD150, CD244, etc., CD150 being the founding member, and, thus, also known as slamF1, i.e., SLAM family member 1.

The signature SLAM codes for the hemopoietic hierarchy are:

For HSCs, CD150 + CD48 − was sufficient instead of CD150 + CD48 − CD244 − because CD48 is a ligand for CD244, and both would be positive only in the activated lineage-restricted progenitors. It seems that this code was more efficient than the more tedious earlier set of the large number of markers, and are also conserved across the mouse strains; however, recent work has shown that this method excludes a large number of HSCs and includes an equally large number of non-stem cells. [9] [10] CD150 + CD48 − gave stem cell purity comparable to Thy1 lo SCA-1 +lin− c-kit + in mice. [11]

LT-HSC/ST-HSC/early MPP/late MPP

Irving Weissman's group at Stanford University was the first to isolate mouse hematopoietic stem cells in 1986 [12] [13] and was also the first to work out the markers to distinguish the mouse long-term (LT-HSC) and short-term (ST-HSC) hematopoietic stem cells (self-renew-capable), and the Multi-potent progenitors (MPP, low or no self-renew capability – the later the developmental stage of MPP, the lesser the self-renewal ability and the more of some of the markers like CD4 and CD135):

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KSL cells in cell biology are an early form of mouse/murine hematopoietic stem cells. Characteristics are Kit (+), Sca-1 (+) and Lin (-). HSCs [Hematopoietic stem cells] in murine cultures show phenotypic markers as being CD34-, CD150+, and Flt3- for LTR [long-term reconstitution]. These phenotypic markers are used when purifying hematopoietic stem cells from the many other differentiated cells in the bone marrow.

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References

  1. ↑ Civin CI. Strauss LC. Brovall C. Fackler MJ. Schwartz JF. Shaper JH. (1984). "Antigenic analysis of haematopoiesis:III. hematopoietic cell surface antigen defined by a monoclonal antibody raised against KG-1a cells". Journal of Immunology. 133 (1): 157–65. doi:10.4049/jimmunol.133.1.157.
  2. ↑ Tindle RW, Nichols RA, Chan L, Campana D, Catovsky D, Birnie GD (1985). "A novel monoclonal antibody BI-3C5 recognises myeloblasts and non-B non-T lymphoblasts in acute leukaemias and CGL blast crises, and reacts with immature cells in normal bone marrow". Leukemia Research. 9 (1): 1–9. doi:10.1016/0145-2126(85)90016-5. PMID   3857402.
  3. ↑ Tindle RW, Katz F, Martin H, Watt S, Catovsky D, Janossy G, Greaves M (1987). "BI-3C5 (CD34) defines multipotential and lineage restricted progenitor cells and their leukaemic counterparts". Leucocyte Typing III: White Cell Differentiation Antigens: 654–55.
  4. ↑ Loken MR, Shah VO, Civin CI (1987). "Characterization of myeloid antigens on human bone marrow using multicolour immunofluorescence". Leucocyte Typing III: White Cell Differentiation Antigens. pp. 630–35.
  5. ↑ "Stemcells Redirection". stemcells.nih.gov. Archived from the original on 2011-05-25. Retrieved 2019-07-08.
  6. ↑ Bhatia M, Bonnet D, Murdoch B, Gan OI, Dick JE (September 1998). "A newly discovered class of human hematopoietic cells with SCID-repopulating activity". Nature Medicine. 4 (9): 1038–45. doi:10.1038/2023. PMID   9734397.
  7. ↑ Guo Y, Lübbert M, Engelhardt M (2003). "CD34- hematopoietic stem cells: current concepts and controversies". Stem Cells. 21 (1): 15–20. doi: 10.1634/stemcells.21-1-15 . PMID   12529547.
  8. ↑ Doi H, Inaba M, Yamamoto Y, Taketani S, Mori SI, Sugihara A, Ogata H, Toki J, Hisha H, Inaba K, Sogo S, Adachi M, Matsuda T, Good RA, Ikehara S (March 1997). "Pluripotent hemopoietic stem cells are c-kit<low". Proceedings of the National Academy of Sciences of the United States of America. 94 (6): 2513–7. Bibcode:1997PNAS...94.2513D. doi: 10.1073/pnas.94.6.2513 . PMC   20119 . PMID   9122226.
  9. ↑ Weksberg DC, Chambers SM, Boles NC, Goodell MA (February 2008). "CD150- side population cells represent a functionally distinct population of long-term hematopoietic stem cells". Blood. 111 (4): 2444–51. doi:10.1182/blood-2007-09-115006. PMC   2234069 . PMID   18055867.
  10. ↑ Van Zant G (2005). "Stem cell markers: less is more!". Blood. 107 (3): 855–56. doi: 10.1182/blood-2005-11-4400 .
  11. ↑ Kiel MJ, Yilmaz OH, Iwashita T, Yilmaz OH, Terhorst C, Morrison SJ (July 2005). "SLAM family receptors distinguish hematopoietic stem and progenitor cells and reveal endothelial niches for stem cells". Cell. 121 (7): 1109–21. doi: 10.1016/j.cell.2005.05.026 . PMID   15989959.
  12. ↑ Muller-Sieburg, Christa E; Whitlock, Cheryl A; Weissman, Irving L (1986). "Isolation of two early B lymphocyte progenitors from mouse marrow: A committed Pre-Pre-B cell and a clonogenic Thy-1lo hematopoietic stem cell". Cell. 44 (4): 653–662. doi:10.1016/0092-8674(86)90274-6. PMID   2868799.
  13. ↑ Weissman, Irving L; Shizuru, Judith A (2008). "The origins of the identification and isolation of hematopoietic stem cells, and their capability to induce donor-specific transplantation tolerance and treat autoimmune diseases". Blood. 112 (9): 3543–3553. doi:10.1182/blood-2008-08-078220. PMC   2574516 . PMID   18948588 – via ASH Publications.
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