methods in heamatopoietic stem cell assays - part -2

Heamatopoietic stem cell assays


I Surrogate short-term in vivo and in vitro assays for detecting HSCs and their progeny:

I) Immunophenotypical Analysis of HSC / Progenitors
- This method relay on fluorescence –activated cell sorting (FACS)-based methods. The most commonly used FACS-purified populations of HSC/progenitors cells include the following:
ia)- Thy1.1(10), Lin- Sca1+Cells:– is for isolation of short term repopulating HSCs and based on expression of their stem-cell antigen (scs-1-, low expression of Thy1.1 and lack of expression of lineage markers
ib) Lin-c-Kit+ Sca-1+ Cells (LKS+)
- in recent advance, expression of CD34 and Flt3(CD135) has been used to further purify long-term repopulating HSC ( LKS+ CD34- Flt-3-) from short-term repopulating HSCs( LKS+CD34+ Flt-3-) and multipotent progenitors ( LKS+ CD34+Flt-3+)

ii) Fluorescent Dyes on High Drug Efflux Properties of HSCs: Rhodamine 123, Hoescht 33342, and the side population:

here two different dyes mitochondrial binding dye Rhodamine 123 (Rh123) and DNA-binding dye Hoescht 33342 (Ho 33342) used either alone or combination for isolation of HSC; and isolation of Hoescht 33342 (Ho 33342) side population (SP), which emits Ho 33342 at two wavelengths simultaneously, resulting in a distinct ‘tail ‘profile, which disappears with the drug Verapamil (the drug used for blocking the activity of ATP-binding cassettes (ABC) transporters superfamily.

iii) SLAM Family Members: SLAM proteins are a family of cell surface glycoproteins in the immunoglobulin superfamily with specific SLAM antigen (CD150+ CD244-CD48-), which is also applying to purify a population of which approximately 50% of single cells reconstituted lethally irradiated animals.
- this SLAM method more useful to detect HSCs in older, mobilised, or transplanted mice,


II
In vitro and Short-term in Vivo Assays for detecting Functional Potential of HSCs and Progenitors:

This method often sued for measuring the mature progenitors,
i) Colony –Forming Cell or CFU mixed Assays:
- the Colony –Forming Cell(CFC) assay measures the progenitor cells in a give n population using semisolid agar- or more commonly, well defined methylcellulose –based culture media,
- the majority of CFCs consists of lineage restricted colonises such as BFU-E, CFU-G, CFU-GM, etc,
- the most immature (multipotent) CFC measurable contains Granulocytes, Erythrocytes, Macropages, and often Megakaryocytes (CFU-GEMM),
- unfortunately ,t his CFC method do not measure the HSCs cells

ii) Cobblestone Area-Forming Cells (CAFC) /Long-term Culture Initiating Cells(LTC-IC):
- It’s a coculture system, used to measure the HSCs frequencies,

iii) Short-term in vivo Assays:
- the cells which is accountability is Colony-forming unit- spleen (CFU-S),
- this cells once injected into an irradiated mice recipient, hoe to spleen and form macroscopic colonies that provide very short term (usually 1-3 weeks ) in vivo repopulation of the mouse,

III) In vivo assays to measure HSC numbers and their Functional potential:

-this assays, in general, is focusing on the long-term repopulating assays, and the most common one is Competitive Repopulating Assay (CRA). This CRA measures the functional potential of an unknown source of HSCs against a set known numbers of HSCs (usually whole bone marrow cells from the congenic wilt-type mice),
- this method also provide qualitative or semi quantitative information about the HSCs within a given population but it can’t distinguish between the numbers of HSCs o their quality (progeny produced peer HSC),


- while competitive repopulating units (CRU), which uses minimal number of HSCS, measure the quantity of HSCs, repopulating units (RU) measures the functional quality of HSCs.